Multiparameter analysis for drug response and related methods

ABSTRACT

The invention provides a method of determining a comparative expression profile in an individual by comparing the expression levels of a sample of molecules in a population of molecules in a specimen from the individual with a health-associated reference expression region of the sample of molecules, wherein expression levels within the health-associated reference expression region indicate a reference expression profile and wherein expression levels outside the health-associated reference expression region indicate a perturbed expression profile. The invention also provides methods of diagnosing a disease or a health state in an individual by comparing the expression level of a sample of molecules in a specimen from the individual with a health-associated reference expression region of the sample of molecules. The invention additionally provides a method of classifying a population by drug responsiveness.

BACKGROUND OF THE INVENTION

The present invention relates generally to methods of predictivemedicine and more specifically to methods of determining expressionprofiles of an individual in response to a drug.

Every living organism utilizes genetic information in the form ofdiscrete nucleotide sequences, called genes, to convey information forthe proper development and function of the organism. Even simpleorganisms, such as bacteria, contain thousands of genes, and the numberis many fold greater in complex organisms such as humans. Understandingthe complexities of the development and functioning of living organismsrequires knowledge of these genes.

For many years, scientists have searched for and identified a number ofgenes important in the development and function of living organisms.What was once a difficult and time consuming process has greatlyaccelerated in recent years due to advances in technology and directedprojects aimed at identifying essentially all genetic information of anorganism. The first draft of the human genome is now available, and morethan 30 organisms have now had their entire genomes sequenced. Thedetermination of the genome of additional organisms is currently beingpursued.

One of the most ambitious of these genomic projects has been the HumanGenome Project, with the goal of sequencing the entire human genome. Thevast amount of genetic information available from the Human GenomeProject provides a rich resource of potential targets for drug discoveryas well as new diagnostic tools for medicine.

Although the determination of essentially all genes expressed in anorganism is a rich resource of information, there remains the dauntingtask of applying this knowledge in a manner that is useful for practicalmedical applications. Perhaps 50,000 genes are expressed in human, andthe analysis of such a large number of genes is complex. Moreover, inaddition to the large number of genes, another layer of complexityarises from alternative splicing of mRNA and various modifications ofproteins encoded by the genes. Furthermore, these gene expressionpatterns are expected to change when a individual has a disease.Information on gene expression patterns thus provides a basis forefficient and accurate diagnostic methods based on changes in geneexpression in various diseases. The exploitation of genomics andproteomics information thus requires methods that can account for thelarge number of genes and complexity of gene expression patterns usefulfor medical applications. Fully exploiting genomics and proteomicsinformation for medical applications requires methods that canaccurately and efficiently monitor complex changes in gene expressionpatterns both at the mRNA and protein levels.

Thus, there exists a need for methods to efficiently diagnose a diseasebased on gene expression patterns in an individual. The presentinvention satisfies this need and provides related advantages as well.

SUMMARY OF THE INVENTION

The invention provides a method of determining a comparative expressionprofile in an individual by comparing the expression levels of a sampleof molecules in a population of molecules in a specimen from theindividual with a health-associated reference expression region of thesample of molecules, wherein expression levels within thehealth-associated reference expression region indicate a referenceexpression profile and wherein expression levels outside thehealth-associated reference expression region indicate a perturbedexpression profile. The invention also provides methods of diagnosing adisease or a health state in an individual by comparing the expressionlevel of a sample of molecules in a specimen from the individual with ahealth-associated reference expression region of the sample ofmolecules. The invention additionally provides a method of classifying apopulation by drug responsiveness.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic diagram of a hypothetical health-associatedreference expression region. The circles represent multidimensionalcoordinate points representative of the expression levels of twomolecules in an individual. The expression levels are in arbitraryunits. The top and bottom panels show a health-associated referenceexpression region of reference individuals in two-dimensional space as aregion of coordinate points. The panels also show the coordinate pointsof two individuals that lie outside the health-associated referenceexpression region.

FIG. 2 shows a schematic diagram of a hypothetical health-associatedreference expression region. The circles represent multidimensionalcoordinate points representative of the expression levels (in arbitraryunits) of three molecules in an individual. Shown is a health-associatedreference expression region of reference individuals inthree-dimensional space as a region of coordinate points and thecoordinate point of an individual that lies outside thehealth-associated reference expression region.

FIG. 3 shows the coordinate points in two-dimensional spacerepresentative of the expression levels (in arbitrary units) of twomolecules. The data set shows three health states that can be classifiedin three regions, corresponding to three health-associated referenceexpression regions.

FIGS. 4A and B shows a data set for three health states and twomolecular expression levels determined by logistic regression analysis,with FIG. 4B showing the coordinates of individuals “A” (×) and “B” (+).

FIG. 5 shows a data set for three health states and two expressionlevels determined by machine learning by boosting of individualmolecules.

FIG. 6 shows a flow diagram that describes the operation of a method ofdetermining a comparative expression profile one molecule at a time.

FIG. 7 shows a flow diagram that describes the operation of a method ofdetermining a comparative expression profile in a multidimensionalanalysis.

FIG. 8 shows a block diagram of a computer system containing acomparative expression profiler.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a method of determining a comparative expressionprofile in an individual by comparing the expression levels of a sampleof molecules in a population of molecules in a specimen from anindividual with one or more health-associated reference expressionregions of the sample of molecules. The specimen molecules can benucleic acids, polypeptides, or small molecules.

The methods of the invention use statistically determinedhealth-associated reference expression regions representing theexpression levels of a sample of molecules in a population of referenceindividuals having a selected health state. For example, referenceindividuals can be normal, healthy individuals, and the expressionlevels in a population of healthy individuals can be determined forvarious molecules.

The methods of the invention can be used in a multiparameter analysis bymeasuring the expression levels of multiple molecules representative ofthe health state of an individual. For example, the expression levels ofa sample of molecules in a specimen from an individual can be comparedto a health-associated reference expression region representing theexpression ranges of the corresponding individual molecules determinedfor the reference population of healthy individuals in aone-molecule-at-a-time analysis. In addition, the expression levels ofthe sample of molecules can be compared to the other molecules of thesample of molecules and to one or more health-associated referenceexpression regions in a multidimensional analysis. Such a comparison isuseful for determining whether an individual has a health state similarto that of the reference population, for example, a healthy individual,or a health state that deviates from the reference population, forexample, a disease state. The methods of the invention can also be usedto classify a population by drug responsiveness, as well as predict adrug response in an individual, for example, in pharmacokineticsapplications. Thus, the methods of the invention can be used todetermine the health state or drug responsiveness of an individual incomparison to a reference population and are particularly useful fordiagnostic applications for human individuals compared to humanpopulations.

Expression levels of the specimen molecules that are within ahealth-associated reference expression region indicate a referenceexpression profile, whereas expression levels outside thehealth-associated reference expression region indicate a perturbedexpression profile. The methods of the invention are advantageous inthat they can be used to predict the health state of an individual bydetermining whether the individual has a reference expression profileindicative of a reference health state or a perturbed expression profileindicative of a potential disease state in the individual or of apredisposition to developing a disease. Moreover, the methods of theinvention provide a multiparameter analysis of an individual'sexpression profile by measuring the expression level of multiplemolecules, thus allowing the determination of an expression profile thatis predictive of an individual's health, including the diagnosis of adisease, the prognosis of a disease, or estimating the course of adisease.

An individual who has a disease or is in early stages of developing adisease has characteristic changes in expression of molecules in a cell,including changes in gene expression that affect mRNA and proteinexpression, changes in modifications of molecules expressed in a cell,and/or changes in the expression of small molecules expressed in a cellor fluid sample from an individual. Changes in expression of moleculescan reflect a disease state or a predisposition to developing a disease.Monitoring the expression level of molecules in a cell can thus be usedto generate an expression profile, which can be correlated with thehealth of an individual. Such an expression profile is essentially asnapshot of the physiological state of the individual.

Although a particular disease can primarily affect one or a few systems,for example, cardiovascular disease affecting primarily thecardiovascular system, it is expected that a relatively homogeneouspopulation of cells can provide a representative sampling of cellsreflective of a variety of physiological systems, even if those cellsare not directly associated with the particular disease. One suchrelatively homogeneous population of cells representative of a varietyof physiological systems is white blood cells (WBCs), or subpopulationsthereof. Accordingly, the methods of the invention can be convenientlyperformed with a specimen from an individual such as WBCs, which arereadily accessible and can provide a window into many physiologicalsystems, including cardiovascular, nervous, immune, gastrointestinal,endocrine, hepatic, lymphatic, neuromuscular, renal, respiratory,skeletal, and urogenital, metabolic systems, and the like, as disclosedherein.

For example, although a renal disease can affect primarily cells of therenal system, it is expected that WBCs, which are not directly involvedin the renal disease, will nevertheless provide a window for observingphysiological changes associated with the renal disease. The use of WBCsto monitor a variety of physiological changes is advantageous in that itobviates the need to obtain tissue specimens directly affected by thedisease. Instead, readily accessible WBCs are used.

Furthermore, some white blood cells migrate through tissue and expanddue to abnormalities such as inflammation, diseases such as cancer,autoimmune disease, or any disease that results in an immune responseinvolving white blood cells. Expression of physiologically relevantgenes in WBCs can be reset by control mechanisms in response to variouspathologies. Accordingly, WBCs provide a conveniently accessiblemonitoring system for various pathologies and can therefore beadvantageously used in methods of the invention for diagnosing a diseaseor predisposition to develop a disease, determining the prognosis of adisease, or estimating the course of a disease. The course of a diseaseincludes the stage or severity of the disease, and can include theresponse of a patient to one or more treatments.

For example, macrophages, a subpopulation of white blood cells, respondto physiological changes, which in turn results in biochemical changesin the macrophages. Accordingly, macrophages can function as a windowinto the physiological changes that occur when an individual has adisease, a predisposition to developing a disease, or exhibits aparticular course of a disease. Therefore, macrophages, or other WBCs orsubpopulations thereof, provide a window into observing the network ofphysiological changes that can occur at various stages of diseasedevelopment, including a pre-disease state indicative of apredisposition to developing a disease.

The methods of the invention can be used to diagnose a disease,determine the prognosis of a disease, or predict the course of a diseaseby obtaining a specimen from an individual, which can be a specimen thatincludes WBCs, and determining the health state of the individual.Exemplary diseases include, for example, cancer, including breast,prostate, ovarian, lung colorectal, hepatic, renal, leukemia, andlymphoma; cardiovascular diseases, including heart failure, hypertensionand atherosclerosis; respiratory diseases; renal diseases;gastrointestinal diseases, including inflammatory bowel diseases such asCrohn's disease and ulcerative colitis; hepatic, gallbladder and bileduct diseases, including hepatitis and cirrhosis; hematologic diseases;metabolic diseases; endocrine and reproductive diseases, includingdiabetes; bone and bone mineral metabolism diseases; immune systemdiseases, including autoimmune diseases such as rheumatoid arthritis,lupus erythematosus, and other autoimmune diseases; musculoskeletal andconnective tissue diseases, including arthritis; infectious diseases;and neurological diseases.

In addition, the methods of the invention directed to multiparameteranalysis can also be used to identify one or more genetic defects. Themethods of the invention can be particularly useful for diagnosingdiseases resulting from multiple genetic defects and/or environmentalfactors. Accordingly, the methods of the invention can be useful in thediagnosis of polygenic diseases resulting from mutations in multiplegenetic loci. Furthermore, a combination of genetic defects can bedetermined by methods of the invention, for example, a particularconfiguration of expression profiles can indicate the likely combinationof genetic defects. Such information can be used to stratify a diseaseand can also be used to determine the stage of progression of a disease.

Furthermore, the relatively homogeneous population of WBCs can befurther fractionated, for example, into lymphocytes such as T cells or Bcells, granulocytes, monocytes, macrophages, neutrophils, eosinophils,basophils, mast cells, and the like, and still be used as arepresentative sampling of cells useful for monitoring a variety ofphysiological systems. Even a single cell can be used as arepresentative specimen from an individual for use in methods of theinvention.

The methods of the invention advantageously use a statistical analysisof the expression levels of molecules in a reference population ofindividuals to predetermine a health-associated reference expressionregion of molecules as they vary in the reference population. Such ahealth-associated reference expression region can be used to compare theexpression level of molecules in an individual as a diagnostic method todetermine the health state of the individual. The expression profile ofan individual can be correlated with the health state of an individual,including whether an individual is healthy, has a disease, or has apredisposition to developing a disease. Such an expression profile isalso useful in prognostic applications, including determining theprognosis of an individual who has a disease, selecting a therapy thatis tailored to the physiological or genetic state of the individual, orestimating the course of a disease. Such information on the expressionprofile of an individual is thus applicable in both predictive medicineand preventive medicine.

The methods of the invention can be used as a tool for predictivemedicine to diagnose a disease or diagnose the health state of anindividual. Variations in expression of molecules such as DNA, mRNA,polypeptides or small molecules can be used to predict the health stateof an individual. For example, an individual having expression levels ofmolecules that fall within a health-associated reference expressionregion is predicted to have a health state similar to the referencepopulation. In the case where the reference population is healthyindividuals, the individual is diagnosed as being healthy. An individualhaving molecules with expression levels outside the health-associatedreference region has a perturbed health state, which can be correlatedwith a particular disease.

The methods of the invention can also be used to predict apredisposition to developing a disease or progression of a disease, forexample, whether the disease is at an early stage or a late stage, bydetermining the expression levels of molecules that correlate withprogress of the disease. Changes in expression levels of certainmolecules are expected to occur during progression of a disease, andsuch changes in expression can therefore be used to predict the progressof a disease or a predisposition to developing a disease. Furthermore,once a correlation between expression levels and disease progression hasbeen made, the methods of the invention can be used in preventivemedicine by monitoring an individual for changes in expression levelsthat correlate with a predisposition to developing a disease or earlystages of a disease. The individual can be then be treatedprophylactically to prevent developing a disease or progression to amore severe form of the disease.

The methods of the invention use a statistically determinedhealth-associated reference expression region of molecules indicative ofexpression levels of molecules in a population of reference individualshaving a selected health state, thus accounting for natural variation inthe expression of molecules in a population of reference individuals.The expression levels of molecules in a specimen from an individual canbe compared to the statistically determined health-associated referenceexpression region to determine a comparative expression profile of theindividual relative to the reference population. The determination ofthe reference expression region of a variety of molecules provides abasis for comparing any individual to determine if the individual hasone or more molecules with aberrant expression or molecules havingaberrant relative expression. Thus, the determination of ahealth-associated reference expression region for any number ofmolecules expressed in a cell provides a central repository ofinformation, which can be accessed by a variety of means to determine acomparative expression profile of an individual. The analysis of anindividual's expression profile can be advantageously performed using acomputer, allowing direct or remote linking to a central repository ofone or more health-associated reference expression regions generated bymethods disclosed herein.

The methods of the invention can be used in direct diagnostic methodsperformed in a clinical laboratory or physician's office. Alternatively,the methods of the invention can be used in remote diagnostic methods inwhich the step of measuring the expression levels of molecules isphysically separated from the step of comparing expression levels ofmolecules to a health-associated reference expression region. Forexample, the measurement of the expression levels of molecules can beperformed by a health care professional or the patient at a remotelocation, such as a clinical laboratory, physician's office, or anindividual's home, and the comparison step performed at a differentlocation by conveniently interfacing the remote locations via a networksuch as the internet.

The methods of the invention can employ a variety of analytical systemsto measure the expression levels of molecules in a specimen from anindividual to be tested and from reference individuals for determiningthe health-associated reference expression intervals for variousmolecules. One convenient method for determining expression levels ofmolecules is to use a direct quantitation method such as theisotope-coded affinity tag (ICAT) method (Gygi et al., NatureBiotechnol. 17:994-999 (1999)). The ICAT method involves the comparisonof a test sample and reference sample, which are differentially labeledwith isotopes that can be distinguished using mass spectrometry, asdescribed in more detail below. Other methods for measuring expressionlevels of molecules includes methods in which specimen molecules arefirst bound to a target such as an array based method. Molecules in aspecimen from an individual are bound to target ligands on an array anddetected to measure expression levels of the molecules, as describedbelow. In addition to using an ICAT reagent that modifies polypeptidesor fragments thereof having particular amino acids, polypeptideprofiles, for example, a peptide map of a polypeptide where the peptidescan be correlated with the polypeptide, can be used to measure theexpression level of a polypeptide. Use of a peptide map to correlatewith a polypeptide expression level can be used to obviate the labelingrequired for using the ICAT method, if desired.

As used herein, “expression level” refers to the amount of a moleculeexpressed in a cell that corresponds to the physiological state of thecell. The expression level of a molecule can be represented by theamount of messenger RNA (mRNA) encoded by a gene, the amount ofpolypeptide corresponding to a given amino acid sequence encoded by agene, or the amount of biochemical forms of molecules expressed in acell, including the amount of particular post-synthetic modifications ofa molecule such as a polypeptide, nucleic acid or small molecule. Assuch, an expression level is intended to include a “gene expressionlevel,” a “cellular expression level,” or both. The expression level canrefer to an absolute amount of the molecule in a specimen or to arelative amount of the molecule. The expression level of a molecule canbe determined relative to a control molecule in the specimen.

As used herein, “gene expression level” refers to the amount of amolecule encoded by a gene. The gene expression level of a molecule isintended to include the amount of mRNA, which is determined by thetranscriptional activity of the gene encoding the mRNA, and thestability of the mRNA, which is determined by the half life of the mRNA.The gene expression level is also intended to include the amount of apolypeptide corresponding to a given amino acid sequence encoded by agene. Accordingly, the expression level of a gene can correspond to theamount of mRNA transcribed from the gene, the amount of polypeptideencoded by the gene, or both.

As used herein, a “cellular expression level” refers to the amount of abiochemical form of a molecule expressed in a cell. Such differingbiochemical forms are due to post-synthetic changes in the molecule, forexample, processing or splicing of nucleic acids, posttranslationalmodifications of polypeptides, or modifications of small molecules. Suchpost-translational modifications of polypeptides include, for example,phosphorylation, lipidation, prenylation, sulfation, hydroxylation,acetylation, addition of carbohydrate, addition of prosthetic groups orcofactors, formation of disulfide bonds, proteolysis, assembly intomacromolecular complexes, and the like. As such, a molecule such as apolypeptide having a specific amino acid sequence can exist in multiplebiochemical forms, each of which can be quantitated to determine acellular expression level. For example, a cellular expression level of amolecule can be the amount of a particular form of the molecule such asthe phosphorylated form of a polypeptide. Furthermore, multiple forms ofthe molecule can exist, for example, based on the phosphorylation stateat different sites on the same polypeptide. The amount of each of thesedifferent biochemical forms is intended to be included in the meaning ofa cellular expression level. Furthermore, a polypeptide itself can bemeasured for expression levels or, if desired, peptide fragments thatare correlated with a polypeptide, for example, peptides of a peptidemap, can be measured. As such, analysis of a sufficient number ofpeptides to correlate with a polypeptide functions as a polypeptideprofile and can be used to correlate the expression level of apolypeptide molecule.

A biochemical form of a small molecule can include, for example, amodification of a sugar, including glucose or modifications thereof suchas glucose 1-phosphate, glucose 6-phosphate, glucose 1,6-diphosphate,glucuronic acid, glucosamine, N-acetylglucosamine, and the like. Otherexemplary small molecules include other sugars and carbohydrates,including lactose, maltose, galactose, fructose, and xylose, derivativesthereof, and metabolites thereof such as lactate and pyruvate; salts,ions, atoms and metals such as sodium, potassium, chloride, calcium,bicarbonate/CO₂, chromium, iron, magnesium, manganese, phosphate,molybdenum, selenium, zinc, copper, cobolt, fluoride, nickel, vanadium,silicon, arsenic, boron and the like; amino acids; lipids, includingcholesterol, triglyceride and fatty acids; neurotransmitters andmetabolites thereof such as acetylcholine, dopamine, norepinephrine,epinephrine, serotonin, γ-aminobutyrate, metanephrine, normetanephrine,vanillylmandelic acid, 3-methoxy-4-hydroxyphenylglycol, homovanillicacid, 5-hydroxyindoleacetic acid. The small molecules can beintermediates or products of metabolic or synthetic pathways. Changes inthe expression of small molecules occurs in various diseases and can beused to predict a disease or susceptibility to a disease. For example,an iron deficiency is indicative of certain diseases while an ironexcess is indicative of different diseases. Thus, the level of iron inan individual can be used, for example, in combination with othermolecules, including other small molecules, nucleic acids, orpolypeptides, to determine the health state of an individual.

As used herein, an “expression profile” refers to a characteristicrepresentation of the expression level of at least two molecules in aspecimen such as a cell or tissue. The determination of an expressionprofile in a specimen from an individual is representative of theexpression state of the individual. An expression profile reflects thegene expression level and/or cellular expression level of at least twomolecules in a specimen such as a cell or tissue.

An expression profile can be related to the expression levels ofmultiple molecules, allowing multiparameter analysis and correlationwith the health state of an individual. For example, the expressionprofile of an individual will be perturbed by exposure to environmentalor internal stimuli that result in physiological changes such asexposure to compounds or other environmental challenges or internalchanges due to disease or other conditions that alter physiology. Suchchanges in expression can be readily be measured, as disclosed herein,and correlated with the physiological changes. In the case whereparticular molecules exhibit such variation in expression that theycannot be correlated with the corresponding physiological change in theindividual, such molecules can be discarded from the analysis.

As used herein, a “gene expression profile” refers to a characteristicrepresentation of the gene expression level in a specimen such as a cellor tissue. The determination of a gene expression profile in a specimenfrom an individual is representative of the gene expression state of theindividual. A gene expression profile reflects the expression ofmessenger RNA or polypeptide encoded by one or more genes in a cell ortissue.

As used herein, a “cellular expression profile” refers to acharacteristic representation of the cellular expression level in aspecimen such as a cell or tissue. The determination of a cellularexpression profile in a specimen from an individual is representative ofthe cellular expression state of the individual. A cellular expressionprofile reflects the expression levels of biochemical forms of messengerRNA or polypeptides encoded by one or more genes in a cell or tissue, orby small molecules expressed in blood, a cell or tissue. The cellularexpression profile can also reflect ratios of different types of cells.Accordingly, if desired, a specimen can be optionally analyzed in a cellsorter to determine if cell ratios have changed from a referencepopulation. Such a cell sorting analysis can be performed to enrich fora subpopulation of cells, for example, a subpopulation of WBCs isolatedwith ligands specific for cell surface antigens, as disclosed herein. Inaddition, information on cell ratios can be combined with expressionprofiles determined by methods of the invention to provide additionalinformation useful in diagnosing a disease, determining the prognosis ofa disease, or predicting the course of a disease.

As used herein, a “comparative expression profile” refers to anexpression profile that reflects the expression levels of moleculesrelative to a health-associated reference expression region. Acomparative expression profile thus reflects the expression level of twoor more molecules in an individual relative to the reference expressionlevels for the respective molecules, that is, whether the expressionlevel of a molecule is within a health-associated reference expressionregion or whether the expression level of the molecule is outside ahealth-associated reference expression region.

As used herein, a “region,” when used in reference to expression levelsof molecules, refers to a region of multidimensional space classifiedusing one or more statistical methods. The region represents aclassification of expression levels that is representative of a healthstate and is diagnostically useful for determining the health state ofan individual. One or more statistical methods, as disclosed herein, canbe used to define a region of multidimensional space. Exemplarystatistical methods include, for example, discriminant analysis,classification analysis, cluster analysis, analysis of variance (ANOVA),regression analysis, regression trees, decision trees, nearest neighboralgorithms, principal components, factor analysis, multidimensionalscaling and other methods of dimensionality reduction, likelihoodmodels, hypothesis testing, kernel density estimation and othersmoothing techniques, cross-validation and other methods to guardagainst overfitting of the data, the bootstrap and other statisticalresampling techniques, artificial intelligence, including artificialneural networks, machine learning, data mining, and boosting algorithms,and Bayesian analysis using prior probability distributions.

As used herein, a “health-associated reference expression region” refersto a region of multidimensional space that is representative of theexpression levels of a sample of molecules in a population of referenceindividuals. A health-associated reference expression region can be usedin a one-molecule-at-a-time analysis, in which the expression levels ofindividual molecules are compared to the expression levels of thecorresponding molecules in a population of reference individuals. When aone-molecule-at-a-time analysis is applied, the expression level of anindividual molecule is compared to a health-associated referenceexpression region that is a health-associated reference expressioninterval. In multidimensional analysis, the expression levels ofindividual molecules in a sample of molecules can be compared to othermolecules in the sample of molecules to determine a multidimensionalcoordinate point representative of the expression levels of a sample ofmolecules in a population.

As used herein, a “drug response-associated reference expression region”refers to a region of multidimensional space that is representative ofthe expression levels of a sample of molecules in a population ofreference individuals having a substantially similar expression profilein response to a drug. The drug response-associated reference expressionregion can be based on the administration of a single drug toindividuals in a reference population, a combination of two or moredrugs, or a combination of one or more drugs with a non-pharmaceuticaltherapy, including diet, physical therapy, exercise, and the like. Ifdesired, the individuals in the population can be treated to decreasephysiological variability between individuals, for example, by havingindividuals fast and/or rest before collecting a specimen, depending onthe nature of the drug and/or disease being treated. The referenceindividuals administered the drug can be individuals having a disease orcondition for which the drug has a known or suspected therapeuticeffect. The reference individuals can also be a group of relativelyhealthy individuals if the effects of the drug on an expression profileare to be determined separately from the effects of an individual havinga particular disease, if desired.

As used herein, a “multidimensional coordinate point” refers to acoordinate defined by “n” parameters, where n is the number of moleculesin a sample of molecules and each parameter is the level of expressionof a molecule in the sample. Accordingly, a multidimensional coordinatepoint representative of the expression levels of two molecules isdefined by two parameters corresponding to the expression levels of thetwo molecules (see FIG. 1). Similarly, a multidimensional coordinatepoint representative of the expression levels of three molecules isdefined by three parameters corresponding to the expression levels ofthe three molecules (see FIG. 2). A multidimensional coordinate pointrepresentative of the expression levels of n molecules is defined by nparameters corresponding to the expression levels of n molecules.Accordingly, multidimensional coordinate points for a group ofindividuals such as a reference population is found in n-dimensionalshape space. Multidimensional coordinate points are determined for asample of molecules in individuals of a reference population, and themultidimensional coordinate points can be used to determine ahealth-associated reference expression region for the referencepopulation.

As used herein, a “health-associated reference expression interval”refers to a statistically determined range of expression levels of amolecule in a population of molecules such as mRNA, polypeptide, smallmolecules, or biochemical forms of a molecule that is determined bymeasuring the expression level of the molecule in a statisticallyrepresentative population of reference individuals. As used herein, a“reference individual” refers to an individual selected for comparisonusing defined criteria. One skilled in the art can readily determinecriteria suitable for inclusion of an individual as a referenceindividual for a particular application of methods of the invention, asdescribed below. As used herein, a “reference population” refers to agroup of two or more reference individuals.

Any relevant criteria can be used for identifying a suitable referenceindividual for a desired comparison. For example, a reference individualcan be a healthy individual who is in good health and essentiallydisease free. One skilled in the art can readily determine if anindividual is in good health based on subjective feelings of well beingof the individual and objective signs of disease in an individual. Othercriteria can include gender, ethnic background, presence of disease, orany criteria useful for comparing the health state of an individual.

Once reference criteria have been identified, for example, the referencecriteria of healthy individuals, a population of individuals is selectedas reference individuals to determine a health-associated referenceregion of molecules expressed in the individuals. One skilled in the artcan readily determine desired criteria for the reference population andselect individuals fitting the desired criteria. In one embodiment, thereference population is healthy individuals. A particularly usefulreference population can be one or more families having members who arehealthy and have a family history indicating no known genetic diseases.Such a reference population of family members can also be useful fordistinguishing those molecules having a statistically reproducibleexpression interval from those molecules having such variability inexpression that no relevant health-associated reference expressionregion can be determined.

An individual expresses a given molecule at a given level that ischaracteristic of the genotype and physiological state of theindividual, including his or her health state. An individual alsoexpresses a set of molecules at a combination of levels whose jointdistribution is characteristic of the genotype and physiological stateof the individual, including his or her health state. Due to geneticvariation, healthy individuals will express variable levels of a givengene depending on the genotype of each individual. Accordingly, thesevariable expression levels of a given gene in a population ofindividuals correspond to a range of expression levels characteristic ofthe health state of the individuals. Such an expression range can bepredetermined by sampling a sufficient number of reference individualsand determining the corresponding statistically useful health-associatedreference expression intervals found in these individuals.

An individual can also be characterized with respect to a set ofmolecules having a joint distribution characteristic of the genotype andphysiological state of the individual. The expression levels of such aset of molecules can be used to define a multidimensional coordinatepoint, which can be compared to one or more health-associated referenceexpression regions to determine if the individual has a reference healthstate or a perturbed health state.

In addition to genetic variation, the expression level of molecules canalso vary due to the physiological state of the individual. Even inindividuals considered to be healthy, the expression levels of moleculescan vary depending on the individual's physiological state. For example,the expression level of molecules in an individual can vary with diet,drug intake, age, gender, and physiological state such as exercise,resting or sleeping. Therefore, if desired, a reference individual canbe selected based on criteria that account for such variability, forexample, by controlling diet by collecting specimens from individualsafter 12 hours of fasting or restricting drug intake for an appropriateperiod of time prior to obtaining a specimen.

A health-associated reference expression region is a region ofmultidimensional space determined by the expression levels of a sampleof molecules, and the boundaries of the region represent theperturbation limit, outside of which indicates that an individual has aperturbed expression profile that lies outside the statisticalboundaries of the reference population. For example, in aone-molecule-at-a-time analysis, the upper and lower boundaries of ahealth-associated reference expression interval represent theperturbation limit, above or below which indicates that an individualhas perturbed expression of a molecule that lies outside the statisticalboundaries of the reference population. An individual with perturbedexpression of a molecule, with a level of expression that lies outsidethe interval determined from reference individuals, potentially has adisease state. The greater the number of molecules that are expressed atlevels outside a health-associated reference expression interval, thegreater the likelihood that such perturbations are associated with adisease state. Similarly, the greater the deviation of amultidimensional coordinate point of an individual from ahealth-associated reference expression region, the greater thelikelihood that such perturbations are associated with a disease state.

As used herein, a “reference expression level” refers to the expressionlevel of a molecule that is correlated with a health-associatedreference expression interval. One skilled in the art can readilydetermine a reference expression level by determining the expressionlevel of a molecule in a reference specimen relative to ahealth-associated reference expression interval, for example, usingappropriate standards, as described below. A reference expression levelcan be any level suitable for measuring and comparing expression levelsof molecules between different specimens, although the referenceexpression level will generally be within the health-associatedreference expression interval. In one embodiment, the referenceexpression level can be an average of the health-associated referenceexpression interval (see below).

As used herein, a “sample,” when used in reference to molecules in apopulation, refers to a group of molecules in a population havingexpression levels that are predictive of the health state of anindividual. The sample of molecules in the population includes moleculesthat exhibit disease-specific changes in expression as well as moleculeshaving altered expression in a disease but which are not specific to aparticular disease. A sample of molecules can also be a set of moleculeswith expression levels having a joint distribution characteristic of ahealth state of an individual. In such a case, the expression levels ofindividual molecules can fall within a reference expression interval butstill be considered a member of a sample of molecules because therelative expression of molecules is outside a health-associatedreference expression region (see below and FIG. 1). Accordingly, amolecule having an expression level within a health-associated referenceexpression interval can be included in a sample of molecules if theexpression of that molecule relative to another sample molecule can becorrelated with a health state.

A sample of molecules in a population that is predictive of the healthstate of an individual is a group of molecules having statisticallydeterminable expression ranges in a given reference population. As usedherein, a sample of molecules in a population can exclude moleculesexhibiting expression levels that are so variable in a referencepopulation that no statistically useful health-associated referenceexpression interval can be determined. Additionally, a sample ofmolecules, as used herein, can specifically exclude molecules that donot exhibit changes in expression with various health states since suchmolecules would not be predictive of the health state of an individual.

One skilled in the art can readily determine molecules that do notexhibit changes in expression with various health states or that are sovariable in a reference population that no statistically usefulhealth-associated reference expression region can be determined. Forexample, to determine molecules having variable expression levelsunsuitable for obtaining statistically useful health-associatedreference expression region, expression levels of molecules in areference population can be examined for variability, and thoseexhibiting variability in expression insufficient for determining astatistically useful health-associated reference expression region canbe disregarded. A reference population particularly useful fordetermining molecules with variable expression is one containing familymembers such as healthy family members. Due to the similar geneticbackground of family members, such a reference population can be used toidentify molecules having variable expression since a referencepopulation of related, healthy family members is expected to exhibitlimited genetic variability and, therefore, observed variable expressionis likely associated with molecules that exhibit natural variability inexpression, which can be disregarded if the variability precludesobtaining statistically useful expression intervals. Such a referencepopulation can be useful to identify molecules in the same or otherreference populations that have variable expression such that they arepreferably excluded from analysis of an expression profile of a sample.

As used herein, a “reference expression profile” refers to acharacteristic representation of the expression state of a sample ofmolecules in a population of molecules in a specimen that falls within ahealth-associated reference expression region. As such, a referenceexpression profile indicates that the expression levels determined for asample of molecules in a specimen from an individual lie within thepredetermined expression levels for those sample molecules or within ahealth-associated reference expression region. An individual having areference expression profile therefore has a health state substantiallythe same as the reference population.

As used herein, a “perturbed expression profile” refers to acharacteristic representation of the expression state of a sample ofmolecules of a population that falls outside a health-associatedreference expression region. As such, a perturbed expression profileindicates that the expression level determined for the sample moleculeslies outside the health-associated reference expression intervals forthe sample molecules or that multidimensional coordinate pointsrepresentative of the sample of molecules lie outside ahealth-associated reference expression region. An expression level of amolecule that is below a lower perturbation limit or above an upperperturbation limit or multidimensional coordinate points that lieoutside of a health-associated reference expression region indicatesthat an individual potentially has a disease state. The greater thenumber of molecules having levels outside the health-associatedreference expression intervals of a healthy population, that is, aboveor below the perturbation limits, or the further the deviation of themultidimensional coordinate points from a health-associated referenceexpression region, the more likely such an individual has a diseasestate. The determination of a perturbed expression profile can be usefuleven in those individuals in which the perturbed expression profile isnot associated with a disease state since such a perturbed expressionstate can be used as a prognostic indicator for individuals predisposedto developing a disease state.

As used herein, a “health state” refers to the medical condition of anindividual. As used herein, a “reference health state” or “referencestate” refers to the health state of an individual having a referenceexpression profile and is considered to have substantially the same or asimilar health state as a reference population.

As used herein, a “perturbed health state” refers to the health state ofan individual having a perturbed expression profile. Such an individualhaving a perturbed health state therefore has a sample of molecules in apopulation of molecules with expression levels that lie outside thehealth-associated reference expression region for those samplemolecules. It is understood that a person having a perturbed healthstate relative to a healthy reference population can appear to behealthy in that the individual does not present any signs or symptoms ofa disease. However, such a person having a perturbed health state can bepredisposed to developing a disease. An individual having a perturbedhealth state also includes an individual who has a disease state. Asused herein, a “disease state” refers to the health state of anindividual who has a disease or has signs or symptoms associated with adisease. One skilled in the art can readily determine if an individualhas signs or symptoms associated with a particular disease. Moreover,one skilled in the art can also readily determine whether an individualhas signs or symptoms that are recognizable as lying outside thecondition of a healthy individual.

As used herein, the term “specimen” is intended to mean any biologicalfluid, cell, tissue, organ or portion thereof, that includes one or moredifferent molecules such as nucleic acids, polypeptides, or smallmolecules. The specimens used in methods of the invention containnucleic acids, polypeptides, small molecules or biochemical forms ofpolypeptides that are representative of the expression level ofmolecules in the individual. The term includes specimens present in anindividual as well as specimens obtained or derived from the individual.For example, a specimen can be a tissue section obtained by biopsy, orcells that are placed in or adapted to tissue culture. A specimen canalso be a biological fluid specimen such as blood, urine or saliva. Aspecimen can be further fractionated, if desired, to a fractioncontaining particular cell types. For example, a blood specimen can befractionated into serum or into fractions containing particular types ofblood cells such as red blood cells or white blood cells (leukocytes). Aparticularly useful specimen for use in the invention is white bloodcells since these cells can be correlated with a variety ofphysiological states, as disclosed herein. If desired, a specimen can bea combination of specimens from an individual such as a combination of atissue and fluid specimen, and the like.

As used herein, a “target” means a collection of two or more ligands. Atarget of the invention generally contains a collection of ligands thathave characteristics that are useful for determining the expressionlevel of a molecule in a specimen. As used herein, the term “ligand”refers to a molecule that can selectively bind to a molecule in aspecimen. The term selectively means that the binding interaction isdetectable over non-specific interactions by a quantifiable assay. Aligand can be essentially any type of molecule such as a polypeptide,nucleic acid, carbohydrate, lipid, or any organic derived compound.Moreover, derivatives, analogues and mimetic compounds are also intendedto be included within the definition of this term. Those skilled in theart know what is intended by the meaning of the term ligand. Forexample, the target can contain nucleic acids, which can be used todetect the presence and amount of nucleic acid or polypeptide moleculesin a specimen. Similarly, the target can contain antibodies or bindingmolecules, which can be used to detect polypeptides or biochemical formsof polypeptides in a specimen. Generally, a target contains a sufficientnumber of ligands to generate an expression profile representative ofthe expression level of a sample of molecules in a population ofmolecules in a specimen from an individual. A variety of methods can beused to detect binding of specimen molecules to target ligands, asdisclosed herein.

One skilled in the art can readily determine an appropriate number andtype of ligands to include in a target for use in methods of theinvention depending on the desired application. For example, a generaltarget can be used to indicate the general health state of an individualfor a variety of potential health states. Such a general target containsa relatively large number of ligands that provides a sufficient numberof binding sites for a sample of molecules to indicate the health stateof an individual. For example, a relatively large number of ligands canbe about 500 or more ligands, about 1000 or more ligands, about 2000 ormore ligands, about 3000 or more ligands, about 5000 or more ligands, oreven about 10,000 or more ligands. A general target contains a varietyof ligands, at least some of which can bind to a sample of molecules ina population of molecules in a specimen to be predictive of the healthstate of an individual.

A directed target can also be used when an expression profile of anindividual is intended to indicate the health state of an individualwith respect to a particular disease or group of diseases. With adirected target, the target can contain a smaller number of ligandssince the ligands are directed to identifying sample molecules for amore limited number of health states, thereby requiring a smaller sampleof molecules predictive of a particular disease or group of diseases.One skilled in the art can readily determine a sufficient number ofligands to include in a target to sample molecules in a population toindicate the health state of an individual, as described herein.

As used herein, the term “nucleic acid” or “nucleic acid molecule” meansa polynucleotide such as deoxyribonucleic acid (DNA) or ribonucleic acid(RNA) and encompasses both single-stranded and double-stranded nucleicacid as well as an oligonucleotide. Nucleic acids useful in theinvention include genomic DNA, cDNA, mRNA and synthetic oligonucleotidescorresponding thereto and can represent the sense strand, the anti-sensestrand, or both.

As used herein, the term “polypeptide” refers to a peptide orpolypeptide of two or more amino acids. A polypeptide can also bemodified by naturally occurring modifications such as post-translationalmodifications, including phosphorylation, lipidation, prenylation,sulfation, hydroxylation, acetylation, addition of carbohydrate,addition of prosthetic groups or cofactors, formation of disulfidebonds, proteolysis, assembly into macromolecular complexes, and thelike.

A modification of a polypeptide, particularly ligand polypeptides, canalso include non-naturally occurring derivatives, analogues andfunctional mimetics thereof generated by chemical synthesis, providedthat such polypeptide modification displays a similar functionalactivity compared to the parent polypeptide. For example, derivativescan include chemical modifications of the polypeptide such asalkylation, acylation, carbamoylation, iodination, or any modificationthat derivatizes the polypeptide. Such derivatized molecules include,for example, those molecules in which free amino groups have beenderivatized to form amine hydrochlorides, p-toluene sulfonyl groups,carbobenzoxy groups, t-butyloxycarbonyl groups, chloroacetyl groups orformyl groups. Free carboxyl groups can be derivatized to form salts,methyl and ethyl esters or other types of esters or hydrazides. Freehydroxyl groups can be derivatized to form O-acyl or O-alkylderivatives. The imidazole nitrogen of histidine can be derivatized toform N-im-benzylhistidine. Also included as derivatives or analogues arethose polypeptides which contain one or more naturally occurring aminoacid derivatives of the twenty standard amino acids, for example,4-hydroxyproline, 5-hydroxylysine, 3-methylhistidine, homoserine,ornithine or carboxyglutamate, and can include amino acids that are notlinked by peptide bonds.

As used herein, a “summation value” refers to the sum of a given set ofvalues. For example, a “positive summation value” refers to the sum ofnumbers assigned a positive value. Similarly, a “negative summationvalue” refers to the sum of numbers assigned a negative value.

The invention provides a method of determining a comparative expressionprofile in an individual. The method includes the steps of determining amultidimensional coordinate point representative of the expressionlevels of a sample of molecules in a population of molecules in aspecimen from the individual; comparing the multidimensional coordinatepoint to a health-associated reference expression region of the sampleof molecules; and determining if the multidimensional coordinate pointis within or outside the health-associated reference expression region,wherein the multidimensional coordinate point within thehealth-associated reference expression region indicates a referenceexpression profile and wherein the multidimensional coordinate pointoutside the health-associated reference expression region indicates aperturbed expression profile.

The invention also provides a method of determining a comparativeexpression profile in an individual by comparing the expression levelsof a sample of molecules in a population of molecules in a specimen fromthe individual with health-associated reference expression intervals ofthe molecules in the sample, wherein expression levels within thehealth-associated reference expression intervals indicate a referenceexpression profile and wherein expression levels outside thehealth-associated reference expression intervals indicate a perturbedexpression profile. By comparing the expression levels of a sample ofmolecules to a health-associated reference expression interval, it canbe determined whether the expression levels are within or outside thehealth-associated reference expression interval. The method of theinvention can further comprise the step of inputting the expressionlevel of the molecules in a specimen. Additionally, the method canfurther comprise the step of determining the expression levels ofmolecules in the specimen. For example, the expression level of amolecule can be determined by comparing the expression level of themolecule with a reference expression level correlated with ahealth-associated reference expression interval, for example, usingdirect quantitation methods such as ICAT. Also, the expression level ofa molecule can be determined using binding methods by contacting aspecimen with a target.

The invention additionally provides a method of determining acomparative expression profile in an individual by determining theexpression levels of a sample of molecules in a population of moleculesin a specimen from the individual; determining a multidimensionalcoordinate point representative of the expression levels of a sample ofmolecules; and comparing the multidimensional coordinate point with ahealth-associated reference expression region of the molecules in thesample, wherein the multidimensional coordinate point within thehealth-associated reference expression region indicates a referenceexpression profile and wherein the multidimensional coordinate pointoutside the health-associated reference expression region indicates aperturbed expression profile. The method can further include the step ofdetermining an expression profile in an individual by contacting aspecimen from the individual with a target.

The invention also provides a method of determining a comparativeexpression profile in an individual by determining the expression levelsof a sample of molecules in a population of molecules in a specimen fromthe individual; and comparing the expression levels withhealth-associated reference expression intervals of the molecules in thesample, wherein expression levels within the health-associated referenceexpression intervals indicate a reference expression profile and whereinexpression levels outside the health-associated reference expressionintervals indicate a perturbed expression profile.

The invention further provides a method of determining a comparativeexpression profile in an individual by comparing the expression levelsof a sample of molecules in a population of molecules in a specimen fromthe individual with reference expression levels correlated withhealth-associated reference expression intervals of the molecules in thesample, wherein expression levels within the health-associated referenceexpression intervals indicate a reference expression profile and whereinexpression levels outside the health-associated reference expressionintervals indicate a perturbed expression profile. Such methods ofcomparing the expression levels of a sample of molecules in a populationwith reference expression levels can similarly be applied to diagnose adisease or health state in an individual.

The invention additionally provides a method of determining anexpression profile in an individual by contacting a specimen from theindividual with a target; determining the expression levels of a sampleof molecules in a population of molecules in the specimen; and comparingthe expression levels with health-associated reference expressionintervals of the molecules in the sample, wherein expression levelswithin the health-associated reference expression intervals indicate areference expression profile and wherein expression levels outside thehealth-associated reference expression intervals indicate a perturbedexpression profile.

In methods of the invention, a reference expression profile indicates areference health state in the individual. A perturbed expression profileindicates a perturbed health state, that is, a health state that differsfrom the reference population, and can indicate a disease state in theindividual or a predisposition to develop a disease when the referencepopulation is healthy. The methods of the invention can therefore beused to diagnose a disease state or a predisposition to develop adisease, even though the individual has no signs or symptoms associatedwith the disease.

The methods of the invention for determining a comparative expressionprofile in an individual utilize a health-associated referenceexpression region based on a statistical sampling of the expressionlevels of molecules in reference individuals to determine the range ofmolecule expression levels. Determining a reference expression regionfor molecules provides a statistically determined expression profile ofa reference population of individuals that allows comparison of theexpression profile of an individual to determine if his or herexpression profile falls within the range of expression levels ofreference individuals or if the expression level of one or moremolecules deviates from the reference range.

Once a health-associated reference expression interval has beendetermined for a given molecule, a specimen from any individual can beanalyzed with respect to the expression level of that molecule.Similarly, once a health-associated reference expression region has beendetermined for a sample of molecules, a specimen from any individual canbe analyzed with respect to the expression levels of the sample ofmolecules. A multidimensional coordinate point can be determined that isrepresentative of the expression levels of the sample of molecules andcompared to the health-associated reference expression region todetermine if the expression level of that molecule lies within oroutside the health-associated reference expression region and istherefore outside the perturbation limits of the health-associatedreference expression region. Furthermore, the expression level can becompared with the health-associated reference expression interval todetermine if the expression level of that molecule lies within thehealth-associated reference expression interval or lies above or belowthe perturbation limits of the health-associated reference expressioninterval.

The methods of the invention for determining a comparative expressionprofile in an individual can use multiple health-associated referenceexpression regions, where each region corresponds to a referencepopulation of individuals. A multidimensional coordinate point for anindividual that lies within one of these regions can be used to classifythe individual as having the health state corresponding to the referencepopulation of individuals represented by that region.

In addition to determining the health state of an individual, methods ofthe invention can be used to classify individuals in a population basedon their responsiveness to a drug or combination of drugs, as disclosedherein. The invention thus provides a method of classifying a populationby drug responsiveness. The method includes the steps of determining amultidimensional coordinate point representative of the expressionlevels of a sample of molecules in a specimen from individuals in apopulation of individuals administered a drug; and determining a drugresponse-associated reference expression region of a group ofindividuals in the population using the multidimensional coordinatepoints, thereby classifying the group of individuals into a drugresponse reference population.

The methods of the invention for classifying a population by drugresponsiveness can be used to stratify responses to a drug into, forexample, responder categories. Such a stratification can be useful inpredicting the effectiveness of a course of therapy, for example,whether a therapy should continue at the same dose or higher or lowerdoses. By correlating a drug response-associated reference region of apopulation of individuals with a category of drug responsiveness, thedrug responsiveness of an individual can be predicted based on whetherthe individual has a drug response expression profile within aparticular drug response-associated reference region.

The methods of classifying a drug response are advantageous in thatchanges in expression levels of molecules can be manifested prior to anovert display of a drug response or prior to a full display of aresponse to a drug, thereby allowing an early determination as to theeffectiveness of a drug response. Furthermore, as disclosed herein, themethods can be used to subcategorize a response that can be correlatedwith a particular drug response outcome that can similarly be used todetermine the effectiveness of a drug. For example, if an adverse drugresponse is correlated with reference individuals in a drugresponse-associated reference expression region, an individual having amultidimensional coordinate point within the region can be identified atan early stage of treatment and the therapy adjusted, even prior to anovert display of adverse symptoms. An individual having amultidimensional coordinate point within a drug response-associatedreference expression region correlated with a positive drug response canconfirm the effectiveness of continued therapy. Thus, the methods of theinvention can be used to optimize a therapeutic regimen by predictingthe response of an individual to an administered drug.

A particularly useful specimen for measuring a drug response is whiteblood cells. As described herein, white blood cells are particularlyuseful as a specimen from an individual since they are readilyaccessible and provide a window to the physiological state of anindividual, including response to a drug. The drug can be a smallmolecule, a biological, or any molecule known or suspected of having atherapeutic effect.

The individuals in a population used to identify a drugresponse-associated reference expression region can be individuallyassayed for expression of molecules in a specimen, or the specimens fromindividuals can be pooled prior to assay. As disclosed herein, theindividuals, in addition to being administered a particular drug ofinterest, can be treated so that they are in a relatively similarphysiological state, for example, by fasting, resting, exercising, orany desired physiological condition, or are individuals having the samedisease. Thus, the population of individuals can be relativelyhomogeneous. Alternatively, the population can include individuals withmore varied physiological states, that is, not treated similarly such aswith fasting, resting, and the like. This more heterogeneous populationcan be pooled prior to assay, if desired.

Additionally provided is a method of predicting a drug response in anindividual. The method includes the steps of determining amultidimensional coordinate point representative of the expressionlevels of a sample of molecules in a specimen from an individual treatedwith a drug; comparing the multidimensional coordinate point to a drugresponse-associated reference expression region for individuals treatedwith the drug; and determining if the multidimensional coordinate pointform the individual is within or outside the drug response-associatedreference expression region, wherein the multidimensional coordinatepoint within the drug response-associated reference expression regionindicates the individual has a substantially similar response to thedrug as individuals in a drug response reference population used for thedrug response-associated reference expression region. A substantiallysimilar response to the drug can refer to a substantially similarexpression profile as indicated by a corresponding multidimensionalcoordinate point residing within a drug response-associated referenceexpression region such that individuals having expression of moleculeswithin the reference expression region have a substantially similar drugresponse.

A substantially similar response to the drug can also refer toindividuals having overt manifestations or indications associated with adrug response, that is, overt manifestations or indications that can bean objectively determined by a physician, for example, based on signs ofa disease or a test result, or based on subjective symptoms described bythe patient. One skilled in the art can readily determine overtindications useful for determining a response to a drug.

The methods of the invention can be used to determine a drugresponse-associated reference expression region for individualsadministered a drug without previously categorizing the individuals byovert indications of a drug response. Thus, the methods of the inventioncan be used to categorize or subcategorize drug responses based on theexpression levels of molecules without prior knowledge of any overtindications associated with the drug response. If desired, such a drugresponse-associated reference expression region can be correlated withovert indications associated with the drug response such as changes in asign or symptom of a disease. Alternatively, the methods of theinvention can be used with a group of individuals in a populationpreviously categorized with respect to one or more overt indicationsassociated with response to a drug, and the expression levels ofmolecules in the group of individuals determined based on the previouscategorization. The group of individuals in the population can be theentire population or a portion of the population, depending on thesimilarity or diversity of responses of individuals to the drug.

Thus, the invention also provides a method of categorizing drugresponsiveness in a population. The method includes the steps of (a)determining a multidimensional coordinate point representative of theexpression levels of a sample of molecules in specimens from apopulation of individuals treated with a drug; (b) identifying a firstgroup of individuals having a substantially similar response to thedrug; and (c) determining a drug response-associated referenceexpression region of the first group of individuals using themultidimensional coordinate points of the first group of individuals,thereby categorizing the drug responsiveness of the first group ofindividuals. The method can further include the steps of (d) identifyinga second group of individuals having a substantially similar response tothe drug, the drug response in the second group being different than thedrug response of the first group; and (e) determining a drugresponse-associated reference expression region of the second group ofindividuals using the multidimensional coordinate points of the secondgroup of individuals, thereby categorizing the drug responsiveness ofthe second group of individuals. The method can further includeoptionally repeating steps (d) and (e) one or more times for anadditional group of individuals having a substantially similar responseto the drug, the drug response in the additional group of individualsbeing different than the drug response of identified groups.

To obtain a statistical sampling of the expression levels of moleculesin a reference individual, the expression levels of molecules in apopulation of reference individuals are determined by the methodsdisclosed herein. Once the expression levels of molecules in thepopulation are determined, well known statistical analysis can beapplied to provide a statistically useful reference expression region.If needed, the expression levels of additional reference individuals canbe determined and added to the previously determined expression levelsuntil statistically useful reference expression intervals aredetermined. Similarly, multiple reference expression regions can bedetermined from multiple reference populations.

Methods of the invention, for the purpose of determining the healthstate of an individual based upon expression profiles for the individualand for one or more reference populations, can include linear,non-linear, and/or multivariate calculations from fields includingmathematics, statistics, and/or computer science. Such calculations canproceed in two phases: (1) an overall computation involving trainingand/or estimation using data from the reference population(s) and (2) asimpler computation for an individual using the results of phase 1. Theend result of such calculations is to provide one or more qualitative orquantitative indicators of the health state of the individual.

A variety of calculations can be used in the methods of the invention.Exemplary-calculations useful in methods of the invention includediscriminant analysis, in which a new individual is classified fromknown calculations by training with a set of individuals of knownclassification. For example, data from individuals with known healthstates can be used to classify a new individual as having one of theseknown health states. Other exemplary methods include classificationanalysis, which is similar to discriminant analysis, and multiplediscriminant analysis.

Cluster analysis is a collection of methods to find groups in a set ofdata. Cluster analysis can be used to find groups, for example, to groupdisease-associated molecules or to cluster individuals into groups ofdifferent health states. Such a method can be used to identify a sampleof molecules from a larger population of molecules that are associatedwith a disease state or indicative of a particular disease orprogression of a disease.

Analysis of variance (ANOVA) is a general statistical technique usefulfor testing the significance of differences between and among groups.

Regression analysis is a general statistical analysis for predictingbased on observations and can be used, for example, to predict a healthstate. Logistic regression analysis can be used for the purpose ofclassification (see Example II). Regression trees is a predictive methodbased on a tree structure trained from a set of data. The data set canbe based on the expression levels of molecules or combinations ofmolecules. Training is carried out with a series of decisions. Forexample, a first decision can be if a molecule or group of molecules isexpressed at a high or low level. Then a decision can be based on theexpression of another molecule or group of molecules, and so forth. Themethod is data-based and can be used for predicting the relationshipbetween molecule expression levels and health state. Decision trees aresimilar to regression trees, but the emphasis is on making a decision,for example, deciding the health state of an individual. Nearestneighbor algorithms are distance based classification methods to assignthe closest match to an individual and are useful forindividual-to-individual comparison of complex components.

Principal components, factor analysis, multidimensional scaling andother methods of dimensionality reduction are methods to reduce thenumber of combinations of molecules for an effective classification.Likelihood models are methods using statistical data and probabilitymodels to provide optimal use of statistical information, whereapplicable. Likelihood models provide a specific description of thepattern of variation in data and can be used for estimation andhypothesis testing. Hypothesis testing is a formal process of using datato make decisions. Hypothesis testing can be used to test whether amolecule or set of molecules is useful and should be included in agroup. Hypothesis testing can also be used to decide if a pool ofindividuals is significantly different from another pool or group ofindividuals.

Derived variables can be created and used to increase dimensionalitybeyond the number of molecules in order to help a statistical methodachieve an effective classification. For example, interaction termsformed by multiplying the expression levels of selected pairs ofmolecules can be used.

Kernel density estimation and other smoothing techniques are methodsused for the purpose of averaging out or eliminating noise in data orstatistical variation in data. Cross-validation and other methods toguard against overfitting of the data are used in particular to protectagainst over optimism or over extension of data regarding theperformance of a diagnostic system from a body of data. Cross-validationserves to prevent an overly optimistic appearance of the data, forexample, a set of data can appear to be predictive of two distinctgroups, where cross-validation can be used to compensate for an apparentoverly optimistic appearance of the data. For example, if oneobservation is repeatedly omitted from a data set of individuals withknown health states, its classification based on the remaining data canbe used to obtain a more realistic indication of system performance.

The bootstrap and other statistical resampling techniques are methodsused to resample from the data in order to assess the variability of thesystem computed from such data. Artificial intelligence, includingartificial neural networks, machine learning, data mining, and boostingalgorithms can also be used (see Example III). An artificial neuralnetwork is a computational method trained on a training set to make anew classification, for example, a training set of molecules in areference population to classify a new individual. Machine learning is acollection of automated methods in which training can be used to learnwhat distinguishes a group, for example, groups of different healthstates, and is then used to classify an individual into a group. Aboosting algorithm is an example of machine learning and is based ontaking a simple system of classification methods to assemble morecomplex methods. For example, in a boosting algorithm, the expressionlevels of molecules taken one at a time can be analyzed in a particularsequence to generate a more effective method. Data mining is a methodbased on learning and inferring from large bodies of data and is usefulfor understanding how to use a large data set for calculations. Datamining is particularly useful when using large data sets, for example,examining a large number of sample molecules and/or a large referencepopulation.

The methods of the invention can include a statistical calculation ofthe degree of confidence associated with the assignment of an individualto a health state. Accordingly, two individuals can both havemultidimensional coordinate points within a particular health-associatedreference expression region, for example, a region corresponding toreference individuals having cancer, but with different levels ofconfidence for the diagnosis, for example, one individual can have a 98%confidence of the diagnosis while the other individual has an 85%confidence.

Bayesian analysis using prior probability distributions is a method thatuses expert opinion with prior probabilities along with observed data tomake a decision. The method can therefore incorporate expert opinion toaid in decision making based on prior probability distributions.

Any one, or combination of two or more, of the statistical methodsdescribed above, or other statistical methods useful for characterizingthe expression levels of molecules to determine the health state of anindividual, can be used in methods of the invention.

Although the methods of the invention are based on determining theexpression levels of molecules in a reference population, it isunderstood that the identity of the molecules need not be known. Thus,it is not necessary to know the identity of a particular specimenmolecule that binds to a particular ligand, only that a specimenmolecule that binds to a particular ligand has a measurable expressionlevel that can be correlated with the health state of an individual.However, if desired, the identity of molecules having expression levelscorrelated with the health state of an individual can be determined, forexample, using methods like ICAT, as described herein.

The methods of the invention can be applied to determining theexpression profile corresponding to many physiological systems andstates in the cell, for example, nervous, immune, cardiovascular,gastrointestinal, endocrine, hepatic, lymphatic, neuromuscular, renal,respiratory, skeletal, urogenital systems, and the like. Pathologies inthese systems and perturbations in organs of these systems can bedetermined using methods of the invention. Furthermore, pathologies invarious systems can be assessed using WBCs as a specimen from theindividual, as described herein.

The methods of the invention are advantageous in that multipleparameters are analyzed to assess the health state of an individual. Themethods of the invention can be used to analyze at least two and up tomany molecules in a sample of molecules to determine the health state ofan individual. Accordingly, a sample of molecules can contain 2 or more,3 or more, 5 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90or more, 100 or more, 150 or more, or even 200 or more differentmolecules for which an expression level can be determined. Moreover, thesample molecules can contain 300, or more, 400 or more, 500 or more, 700or more, or 1000 or more molecules. A sample can also contain 2000 ormore, 3000 or more, 5000 or more, or even 10,000 or more molecules. Whenanalyzing a sample containing a large number of molecules, theexpression levels of the molecules can be conveniently performed using atarget-based method such as an array. For example, in a specimen of 5000molecules, if the expression level of 500 those molecules is correlatedwith the health of an individual, simultaneous measurement of theexpression levels of those 500 molecules using methods of the inventionprovides information on the health of the individual.

The analysis of multiple parameters provides a convenient method todetermine a comparative expression profile of an individual relative toone or more reference populations. The methods of the invention areuseful for determining a comparative expression profile of an individualand providing a simplified output that allows a convenient analysis ofthe health state of an individual, for example, whether an individual ishealthy, has a predisposition for a disease, or has a disease. Suchmethods can also be applied to determining the prognosis of a patienthaving a disease or to estimate the course of a disease.

The methods of the invention are advantageous in that the use ofmultiple parameters provides information on the expression levels ofmolecules that can be correlated with the health state of an individualby comparing the expression levels to one or more health-associatedreference expression regions. The methods of the invention can beperformed as a one-molecule-at-a-time analysis, where the expressionlevel of individual molecules are compared to a health-associatedreference expression region, including a health-associated referenceexpression interval. In a one-molecule-at-a-time multiparameteranalysis, the analysis can be simplified, for example, by assigning anumerical value, which can be summed to generate a summation value thatreflects the comparison of multiple parameters to a referencepopulation, as described in more detail below. In a multidimensionalmultiparameter analysis, the analysis can be simplified by determining amultidimensional coordinate point that is compared to one or morehealth-associated reference expression regions defined by one or morereference populations, as described in more detail below. In bothone-molecule-at-a-time and multidimensional analysis, the informationobtained on multiple parameters, that is, the expression levels ofmultiple molecules, is preserved and is useful in determining the healthstate of an individual.

The methods of the invention are advantageous in that the expressionlevel in an individual for any number of molecules can be characterizedto determine an expression profile for the individual. The methods ofthe invention can be particularly advantageous when a large number ofdifferent molecules are being analyzed.

Although an individual having a disease or who is predisposed todeveloping a disease will have a change in expression of variousmolecules, not all molecules will necessarily have a change inexpression. Furthermore, there can be a change in relative expressionbetween two or more molecules, even though the expression levels ofindividual molecules are each within a health-associated referenceexpression interval. Therefore, whether a change in expression isobserved depends on which particular molecule is characterized withrespect to expression levels. For example, if the expression level of aparticular molecule is determined, and if a change in expression levelof that molecule or a change in relative expression to another moleculeis associated with a disease or indicates a predisposition to developinga disease, then an expression profile based on determining theexpression level of that molecule will reflect the health state of thatindividual. An individual having an expression level for that particularmolecule within a health-associated reference expression interval isconsidered to have a reference health state, at least with respect tothat particular molecule. In contrast, an individual having anexpression level for that particular molecule outside ahealth-associated reference expression interval is considered to have aperturbed health state with respect to that molecule.

Although some diseases can be characterized, at least in part, by achange in expression of a particular molecule, generally a number ofmolecules exhibit changes in expression or change in relative expressionin an individual having a disease state. Similarly, changes inexpression of multiple molecules are also associated with apredisposition to developing a disease state, although the number ofmolecules having altered expression levels can be lower than in adisease state. Most diseases cannot be characterized by a change in asingle molecule but are characterized by changes in expression of avariety of molecules, many of which can also have changes in expressionin other diseases. For example, some of the molecules exhibiting changesin expression in an individual having a disease will be specific to thedisease. However, other molecules exhibiting changes in expression willnot be disease-specific but will be molecules exhibiting changes inexpression in a variety of conditions. The methods of the invention areadvantageous in allowing analysis of such diseases having complexchanges in expression patterns by determining multiple parametersindicative of the health state of an individual.

Furthermore, even in diseases where mutations in a single genecontributes to a disease, these mutations are often associated with theactivity or function of the gene or gene product but do not necessarilyaffect the expression level of the gene or gene product. For example,loss of p53 function is found in more than 50% of human tumors (Wang,Anticancer Res. 19:4759-4771 (1999); Hollstein et al., Science 253:49-53(1991)). However, many of these loss of function mutations alteractivity of p53 but not the level of p53 expression. Therefore,mutations in p53 that would be indicative of cancer could not bedetermined by the expression level of p53. Nevertheless, mutations inp53 which would lead to cancer would also lead to alterations in theexpression levels of other molecules due to changes in the physiologicalstate in response to the p53 mutations. Therefore, determination of thealtered expression levels of these other molecules can be used todetermine the health state of an individual, even in the absence ofmeasurable changes in expression levels of disease-specific genes orgene products that cause or contribute to the disease such as p53mutations that alter activity but not expression levels of p53.

The methods of the present invention are advantageous in that themethods allow multiparameter analysis of complex changes in expressionpatterns associated with a disease or predisposition for a developing adisease, which can be converted to a simplified output that allowsdetermination of the health state of an individual. In particular, themethods of the invention are applicable to determining whether anindividual has substantially the same health state as a referencepopulation or a perturbed health state, including a disease state orpredisposition to developing a disease. Thus, a series of parameters,based on the expression level of a sample of molecules in a populationof molecules in a specimen, for example, mRNA, proteins or smallmolecules from leukocytes, or proteins or small molecules from serum,can be measured to determine the health state of an individual.

A comparative expression profile of an individual can be determinedbased on comparing the expression level of a molecule in a specimen fromthe individual with a health-associated reference expression interval ofthe molecule. Similarly, a comparative expression profile of anindividual can be determined based on comparing the relative expressionlevels of two or more molecules in a sample, for example, by determininga multidimensional coordinate point representative of the expressionlevels of the molecules, to one or more health-associated referenceexpression regions corresponding to the molecules. Although amultidimensional coordinate point can be determined for amultidimensional analysis, it is understood that the expression levelsof individual molecules can be compared to the health-associatedreference expression region so long as the region reflects the relativeexpression of the sample of molecules in the reference population.

The methods of the invention can be used to characterize a health statebased on any number of sample molecules, including large numbers ofsample molecules. The analysis of large numbers of molecules can beparticularly useful when trying to discriminate between diseases havingsimilar but distinct changes in expression patterns. If desired, anexpression profile based on the determination of the expression levelsof essentially all molecules expressed in a specimen can be determinedso long as the health-associated reference expression region of a sampleof molecules in a population of molecules in the specimen isstatistically useful for predicting the health state of an individual.

For example, in a one-molecule-at-a-time analysis, a numerical value canbe assigned indicating whether the expression level of a particularmolecule in a specimen falls within a health-associated referenceexpression interval corresponding to a statistical sampling of areference population. Assigning a numerical value based on whether anexpression level falls within a health-associated reference expressioninterval or lies outside the perturbation limits of such a referenceexpression interval allows a potentially complex analysis of theexpression profile of many molecules to be conveniently converted to asimplified numerical output that provides insight into the health stateof an individual.

The invention provides additional methods of determining a comparativeexpression profile in an individual. One such method includes the stepsof (a) comparing the expression level of a molecule in a specimen froman individual with a health-associated reference expression interval ofthe molecule; and (b) assigning a value of 0 if the expression level iswithin the health-associated reference expression interval or assigninga positive numerical value if the expression level is outside thehealth-associated reference expression interval, wherein an expressionlevel within the health-associated reference expression intervalindicates a reference expression profile and wherein an expression leveloutside the health-associated reference expression interval indicates aperturbed expression profile. The method can further include repeatingsteps (a) and (b) one or more times.

Accordingly, methods of the invention can include the step of assigninga numerical value depending on whether the expression level of amolecule is within a health-associated reference expression interval forthat molecule or whether the expression level of the molecule is outsidethe health-associated reference expression interval. For example, if theexpression level of a molecule is within a health-associated referenceexpression interval, a value of 0 is assigned to the expression level ofthe molecule in a particular specimen. In such a case, a value of 0indicates that the individual has a reference expression profile, atleast with respect to the particular molecule for which an expressionlevel was determined. Such an analysis can similarly be applied to thedetermination of the expression level of two or more molecules or anynumber of molecules. In the case where the expression level of each ofthe sample of molecules analyzed lies within its correspondinghealth-associated reference expression interval, a value of 0 isassigned to the expression level of each molecule. Accordingly, if 0 isassigned to the expression level of each sample molecule analyzed, theindividual has a reference expression profile. An individual having areference expression profile can have a reference health state, that is,a health state that is substantially the same or similar to a referencepopulation of individuals.

If the expression level of a molecule is outside a health-associatedreference expression interval for that molecule, a positive numericalvalue can be assigned to the expression level of the molecule in aparticular specimen. For example, in a simplified case, the positivenumerical value can be 1. In such a case, a value of 1 indicates thatthe individual has a perturbed expression profile. Such an analysis cansimilarly be applied to the determination of the expression level of twoor more molecules or any number of molecules. For each molecule havingan expression level outside its health-associated reference expressioninterval, a positive numerical value is assigned. In such a case, eachmolecule having an expression level that is below or above theperturbation limits of a health-associated reference expression intervalfor that molecule is assigned a positive numerical value, indicating adeviation from a reference expression range. Accordingly, if a positivenumerical value is assigned to the expression level of one or moremolecules analyzed in a sample of molecules, the individual has aperturbed expression profile. An individual having a perturbedexpression profile indicates that the individual has a perturbed healthstate, and such an individual can have a disease state, a predispositionto developing a disease, a prognosis associated with a disease ortreatment of a disease, and such an indicated perturbed health state canalso be used to estimate the course of a disease.

When the expression level of a relatively small number of molecules isdetermined and compared to corresponding health-associated referenceexpression intervals for each respective molecule, the determination ofwhether the expression level of molecules indicates a referenceexpression profile or a perturbed expression profile is straightforward.However, as the number of molecules analyzed increases, the analysisbecomes more difficult. The methods of the invention in which numericalvalues are assigned to the expression level of a molecule based onwhether the expression level is within or outside a health-associatedreference expression interval provide a simplified output that can beparticularly useful when a large number of molecules are analyzed.

One approach to simplifying the analysis of an expression profile basedon a larger number of molecules is to sum the values assigned to theexpression levels of the molecules to generate a summation value. Forexample, if 100 molecules are analyzed and none deviate from theirrespective health-associated reference expression intervals, thesummation value would be 0, indicative of a reference expressionprofile. On the other hand, if at least one of the molecules analyzedhas a value of at least 1, the summation value would be at least 1.Thus, a summation value of 1 or greater indicates a perturbed expressionprofile. As the number of molecules having expression levels outsidetheir respective health-associated reference expression intervalsincreases, the summation value correspondingly increases. Thus, highersummation values indicate a larger number of molecules having expressionlevels outside reference ranges.

For example, in the simplified exemplary case where the expression levelof 100 molecules is characterized and a positive value of 1 is assignedto those deviating from their respective health-associated referenceexpression intervals, a summation value of 5 indicates that 5 moleculeshave expression levels either higher or lower than the range forreference individuals. Such a summation value of 5 can be indicative ofa disease state or a predisposition to developing a disease. Similarly,a summation value of 10 indicates that 10 molecules have expressionlevels either higher or lower than the range of reference individuals.Thus, the summation value provides a simplified analysis characteristicof the expression profile of the individual.

Moreover, the higher the summation value, the greater the number ofmolecules having expression levels outside the reference range and themore likely that such an individual has a disease. For example, it ispossible that an individual having expression levels of 5 moleculesoutside the reference range indicates that the individual is predisposedto developing a disease. However, it is possible that a person having 10molecules outside the reference range indicates that the individual hasa disease. Thus, the methods of the invention can be used to determinethe health state of an individual, including determining whether anindividual has a disease state or a predisposition to developing adisease. The methods of the invention are thus applicable to determiningthe likelihood of an individual having a predisposition to developing adisease or the likelihood that an individual has a disease.

The methods of the invention can include the step of assigning positivenumerical values, and, in such a case, the value assigned to anyindividual molecule can be weighted depending on the likelihood thatexpression of the molecule outside the health-associated referenceexpression interval is correlated with a disease or predisposition todeveloping a disease. For example, as described above, decrease or lossof p53 activity, including loss of p53 expression, is found in a varietyof cancers (Hollstein et al. supra, 1991). Thus, a decrease or loss ofp53 expression has a high degree of correlation with the development ofcancer. Accordingly, a decrease in expression of p53 to a level belowthe health-associated reference expression interval can be assigned ahigher numerical value, or a weighted value, since its expression iscorrelated with a predisposition to developing cancer or with havingcancer. In contrast, a molecule that has an increase in expression thatis correlated with cancer and with benign conditions can be given alower numerical value.

A method of the invention in which a summation value is generated basedon the assignment of weighted numerical values can provide greatersensitivity in discriminating between conditions. For example, assumethat a change in expression of 10 molecules is associated with a benigncondition and a malignant condition. However, the 10 molecules havingaberrant expression in the benign condition are not identical to the 10molecules having aberrant expression in the malignant condition. As anexample, assume that aberrant expression of 5 molecules is common toboth the benign and malignant conditions. Such molecules can be assigneda relatively low numerical value. The 5 molecules associated with thebenign condition can be given an intermediate value, whereas the 5molecules associated with the malignant condition can be given arelatively high value. In such a case, an individual having the benigncondition can be readily distinguished from an individual having themalignant condition based on the summation value since an individualwith the benign condition would have a lower summation value than anindividual having the malignant condition.

In addition to assigning weighted values based on which particularmolecules have expression levels outside a health-associated referenceexpression interval, weighted values can also be assigned based on therelative amount of expression. Assigning weighted values based onrelative deviation from health-associated reference expression intervalsis particularly useful when the expression of a molecule varies with theseverity of a disease. For example, a level of expression that is justoutside the perturbation limits of a health-associated referenceexpression interval can be assigned a lower value, and a higher valuecan be assigned the further the expression level deviates from theperturbation limits.

Weighting can also be used when there is some knowledge that expressionlevels of a particular molecule is correlated with a condition. Forexample, variable expression levels of insulin receptor appear to becorrelated with the severity of associated disease (Taylor, Diabetes41:1473-1490 (1992)). Patients with leprachaunism have mutations in bothinsulin receptor gene alleles and have an extreme degree of insulinresistance. In contrast, many patients with type A insulin resistancehave mutations in only one allele of the insulin receptor gene.Moreover, patients with type A insulin resistance having mutations inboth alleles of the insulin receptor gene tend to have fastinghyperglycemia and overt diabetes mellitus in contrast to patients withsingle mutant alleles, who tend to have glucose intolerance despitenormal levels of fasting glucose. In such a case, assignment of aweighted value based on the relative decrease in expression level ofinsulin receptor can be used to distinguish various levels of insulinresistance or a predisposition to developing insulin resistance.

Similarly, overexpression of HER2/neu is associated with poor patientoutcome in breast cancer patients (Slamon et al., Science 235:177-182(1987); Slamon et al., Science 244:707-712 (1989)). Assigning a weightedvalue based on the relative increase of HER2/neu expression above theperturbation limit of a health-associated reference expression intervalof HER2/neu can be used as a prognostic indicator of the likelyprogression of the disease. An indication of an expression level of amolecule that is associated with the progression of a disease canfurther be optionally combined with additional prognostic markers havingaltered expression, to adjust the aggressiveness of therapy or determinea particular type of therapy.

Another useful application of weighting is based on a threshold ofchange in expression. For example, it is possible that an increase inexpression of a molecule above the health-associated referenceexpression interval is correlated with a predisposition to developing adisease whereas an increase in expression of a particular magnitude iscorrelated with having the disease. In such a case, a lower weightedvalue can be assigned if the expression level of the molecule exceedsthe perturbation limit of the health-associated reference expressioninterval and a higher weighted value can be assigned if the expressionexceeds the threshold limit associated with the disease. Depending onthe desired diagnostic application, one skilled can determine adesirable level of weighting. Thus, the assignment of weighted valuescan be used to further distinguish the expression profile of individualsto determine the health state of an individual.

The invention additionally provides a method of determining acomparative expression profile in an individual by (a) comparing theexpression level of a molecule in a specimen from the individual with ahealth-associated reference expression interval of the molecule; and (b)assigning a value of 0 if the expression level is within thehealth-associated reference expression interval, assigning a positivenumerical value if the expression level is greater than thehealth-associated reference expression interval, or assigning a negativenumerical value if the expression level is less than thehealth-associated reference expression interval, wherein an expressionlevel within the health-associated reference expression intervalindicates a reference expression profile and wherein an expression leveloutside the health-associated reference expression interval indicates aperturbed expression profile. A method of the invention can furtherinclude repeating steps (a) and (b) one or more times.

Methods of the invention described above include the step of assigning apositive numerical value if the expression level of a molecule isoutside a health-associated reference expression interval. Although suchmethods are useful for determining an expression profile of anindividual, such methods nevertheless provide a simplified output ofinformation. For example, it is possible that a molecule isoverexpressed in certain conditions and underexpressed in otherconditions. Using methods of the invention in which a positive numericalvalue is assigned when the expression level of a molecule is eitherabove or below the perturbation limits of the health-associatedreference expression interval can mask the distinction between a diseasein which the molecule is increased and a disease in which the moleculeis decreased. In contrast, a more detailed expression profile can beobtained using a method that utilizes this expression information.

One approach to obtaining a more detailed expression profile is to usemethods in which information on the relative change in expression levelsis incorporated. The invention thus provides methods in which anexpression level exceeding the upper perturbation limit is assigned apositive numerical value and an expression level less than the lowerperturbation limit is assigned a negative numerical value. As such,information on the relative increase or decrease in expression of amolecule is preserved.

For example, if a molecule has increased expression in one disease anddecreased expression in another disease, these changes can be assignedcorresponding positive and negative numerical values that reflect anincrease or decrease, respectively, in expression outside ahealth-associated reference expression interval. In contrast to themethods in which both the increase and decrease are assigned positivenumerical values, the assignment of positive and negative values thatcorrespond to an increase or decrease in expression outside ahealth-associated reference expression interval preserves additionalinformation that is reflected in the expression profile of anindividual. In the above example, such information can be used todistinguish between the disease in which a molecule has increasedexpression above an upper perturbation limit of a health-associatedreference expression interval and the disease in which a molecule hasdecreased expression below the lower perturbation limit. As with othermethods of the invention, the numerical value assigned can be weighteddepending on the desired application of the methods.

As described above, positive numerical values can be assigned toexpression levels outside a health-associated reference expressioninterval regardless of whether the expression level of a molecule isabove or below the perturbation limit. However, a similar analysis canbe accomplished when positive and negative values are assigned by simplyusing the absolute values of the negative numbers. Thus, the methods ofthe invention can include summing the absolute values of positive andnegative values to generate a summation value. In such a case, asummation value of 1 or greater indicates an individual has a perturbedexpression profile. Similarly, methods of the invention can include theuse of mathematical functions other than the absolute value function.

For simplicity, the methods described above assign positive and negativevalues to reflect an expression level above or below, respectively, theperturbation limits of a health-associated reference expressioninterval. However, it is understood that the methods can use any type ofidentifier that is useful for determining an expression profile,including positive and negative numbers for expression levels below andabove a perturbation limit or letter identifiers. Furthermore,identifiers can be included to reflect categories of molecules that areassociated with specific diseases such as diabetes or cancer. Oneskilled in the art can readily determine the appropriate type of valuesto assign, for example, appropriate weighted numerical values orinclusion of identifiers, depending on the particular application of themethods of the invention. As with one-molecule-at-a-time analysisdescribed above, weighting can also be used in multidimensionalanalysis.

The above-described one-molecule-at-a-time analysis of multipleparameters is directed to preserving information about an individual'shealth state based on the determination of expression levels of a sampleof molecules from a specimen of the individual. In addition, theexpression levels of molecules can also be analyzed in amultidimensional analysis using statistical methods, as disclosed herein(also see Examples I, II and III). Instead of comparing the expressionlevels of individual molecules of a sample to the correspondinghealth-associated reference expression intervals determined for areference population, as in one-molecule-at-a-time analysis, theexpression level of each molecule in the sample is compared to othermolecules in the sample in a multidimensional analysis. The expressionof the sample of molecules in an individual is then compared to one ormore health-associated reference expression regions of the same sampleof molecules from one or more populations of reference individuals.Therefore, a multidimensional analysis can examine the relativeexpression of a sample of molecules, allowing more subtle changes inexpression patterns to be correlated with the health state of anindividual than provided by a one-molecule-at-a-time analysis.

A simplified example of a multidimensional analysis is shown in FIG. 1.FIG. 1 shows a schematic diagram of a hypothetical health-associatedreference expression region. The circles represent multidimensionalcoordinate points representative of the expression levels (in arbitraryunits) of two molecules in an individual. Therefore, each circlerepresents a coordinate point in multidimensional space, in this exampletwo-dimensional space, that is defined by the expression levels of twomolecules in an individual. The elliptical shaped region shows theclustering of expression levels of a reference population into aclassification region, which is determined by applying statisticalmethods as disclosed herein. In this example, a region intwo-dimensional shape space is classified as a health-associatedreference expression region.

In the top panel of FIG. 1, one coordinate lies outside thehealth-associated reference expression region. The individualcorresponding to this coordinate has expression levels of molecules 1and 2 outside the health-associated reference expression intervals forthose molecules, that is, molecule 1 is expressed at a higher level thanin the reference population and molecule 2 is expressed at a lower levelthan the reference population. The determination of the perturbationlimits of the health-associated reference expression region and whetheran individual's coordinate lies within the region can be determinedusing statistical analysis, as disclosed herein.

Multidimensional analysis can provide additional insights into theexpression profile of an individual than would be apparent from aone-molecule-at-a-time analysis of individual molecules. The bottompanel of FIG. 1 shows an individual having a coordinate that liesoutside the health-associated reference expression region. In this case,the expression levels of both molecules 1 and 2 are withinhealth-associated reference expression intervals for the respectivemolecules, that is, molecules 1 and 2 are expressed within the samerange as the reference population. Nevertheless, by comparing the twomolecules relative to each other, a deviation from the health-associatedreference expression region can be observed. Thus, a multidimensionalmultiparameter analysis can reveal more subtle changes in an expressionprofile that can be useful in determining the health state of anindividual.

A multidimensional analysis can be performed with additional parameters.For example, a multidimensional analysis can be performed in threedimensional space (see FIG. 2). FIG. 2 shows a schematic diagram of ahypothetical health-associated reference expression region inthree-dimensional space. In this case, each coordinate point representsthe expression levels of three molecules in an individual, which definea three-dimensional coordinate point. A three-dimensional ellipsoidrepresents a health-associated reference expression region inthree-dimensional shape space. Also shown is an individual havingcoordinate points that lie outside the health-associated referenceexpression region. As described above for two-dimensional analysis,statistical methods are applied to determine the perturbation limits ofthe three-dimensional health-associated reference expression region andto determine whether an individual has expression levels of molecules ora representative multidimensional coordinate point within the region.

In addition to two- and three-dimensional analysis, a similar analysiscan be applied in n-dimensional space, where n is the number ofmolecules in a sample of molecules, that is, the number of moleculessufficient to predict the health state of an individual. In such a case,a health-associated reference expression region is defined inn-dimensional shape space based on the n-dimensional coordinate pointsof a reference population of individuals. Again, statistical methods areapplied in multidimensional analysis to determine the perturbationlimits of the n-dimensional health-associated reference expressionregion and to determine whether an n-dimensional coordinate point of anindividual is within or outside the region.

The methods of the invention using multiparameter analysis areparticularly useful for analyzing larger numbers of molecules in asample and can provide insights into the expression profile of anindividual that are not revealed when using a one-molecule-at-a-timeanalysis. Another advantage of multidimensional analysis is that theexpression levels of molecules need not be compared to the same type ofmolecule but, instead, can be compared to any type of molecule that isexpressed in an individual and can be correlated with the health stateof an individual. Therefore, the expression levels of nucleic acids canbe compared to the expression levels of polypeptides in amultidimensional analysis, with each molecule compared to othermolecules in the sample. Similarly, nucleic acids or proteins can becompared to small molecules, or nucleic acids, proteins and smallmolecules can be compared relative to each other. Essentially any typeof specimen molecules can be used, alone or in combination with othertypes of molecules, as a sample of molecules to determine the healthstate of an individual. Since there can be a discordance between mRNAexpression and expression of the corresponding encoded protein, such acomparison between different types of specimen molecules can be usefulfor monitoring changes associated with the health state of anindividual.

In an individual having a predisposition to developing a disease, or whois in early stages of a disease, the individual often will exhibit nosigns or symptoms associated with the disease. For example, in earlystages of cancer, an individual can feel healthy. Early detection ofdiseases such as cancer or determining an individual's susceptibility toa disease can be useful for treating an individual prophylactically,before signs or symptoms of the disease develop. An individual having apredisposition to developing a disease or who is in early stages of adisease can exhibit more subtle changes in expression than an individualexhibiting more overt symptoms of a disease. Multidimensionalmultiparameter analysis can be particularly useful in identifying moresubtle changes in expression of molecules associated with early stagesof disease and can therefore be used advantageously in preventativemedicine.

The methods of the invention are advantageous in that an expressionprofile can be analyzed to determine the health state of an individual.Such methods are useful for routine health screening to determine if anindividual has a reference health state, particularly if the referenceindividuals are healthy, or perturbed health state that requires furthermedical analysis or monitoring or that indicates a particular disease ora predisposition to develop a particular disease. Thus, the methods ofthe invention are useful in a variety of applications for predictivemedicine and preventive medicine.

The methods of the invention are based on obtaining a health-associatedreference expression region of a group of any number of molecules thatare useful in diagnostic applications for determining the health stateof an individual. A health-associated reference expression region isdetermined by obtaining information on the expression levels of a groupof molecules in a population of reference individuals. One skilled inthe art can readily determine the number of individuals to be includedin a population to obtain a statistically useful health-associatedreference expression region, as disclosed herein (see, for example,Anderson, An Introduction to Multivariate Statistical Analysis, seconded., section 6.7, Wiley, New York (1984); Tietz Textbook of ClinicalChemistry, 3rd ed., Burtis and Ashwood, eds., W.B. Saunders Co.,Philadelphia, Chapters 11-14, pp. 265-355 (1999)). For example, once theexpression levels of a sample of molecules have been determined based ona given sized population, one skilled in the art can determine if thepopulation size is sufficient for use in methods of the invention byapplying any of a number of statistical methods to the determinedhealth-associated reference expression region and assessing theusefulness of the health-associated reference expression region forpredicting the health state of an individual (see Example I). Using suchstatistical methods allows a prediction of the statistical usefulness ofa health-associated reference expression region for use in methods ofthe invention.

The number of individuals to include in a population for determining ahealth-associated reference expression region can vary depending on theparticular application. For example, if a particular molecule is foundto have a narrow range of expression variability in a referencepopulation, a health-associated reference expression region for thatmolecule or for that molecule relative to another sample molecule can beobtained with a smaller population. In contrast, if a particularmolecule is found to have a wide range of expression variability in areference population, a larger population can be used for thestatistical analysis to determine a health-associated referenceexpression region.

In a method of the invention in which the expression levels of two ormore molecules are determined and compared to a health-associatedreference expression region, each health-associated reference expressioninterval for each molecule need not be determined with an identicalpopulation. The health-associated reference expression interval for eachmolecule is based on a number of individuals in a population sufficientto make a statistically useful determination of the health-associatedreference expression interval, although larger populations can beincluded.

A reference population can be selected on a variety of criteria based onthe particular application of methods of the invention. Exemplarycriteria for selection of reference individuals include the health statesuch as healthy individuals or individuals having a particular disease,age, gender, ethnic background, drug use, alcohol consumption or othercriteria. Thus, if desired, a reference population can be focused onparticular criteria. Alternatively, the reference population can containa variety of individuals having various physiological states, but thereference population is partitioned into subgroups (see Solberg, TietzTextbook of Clinical Chemistry, 3rd ed., Burtis and Ashwood, eds., W.B.Saunders, Philadelphia, Chapter 14, pp. 336-355 (1999)).

One skilled in the art can readily determine an appropriate referencepopulation based on the particular application of methods of theinvention. The methods of the invention use health-associated referenceexpression regions for comparison to the expression levels of a sampleof molecules in a specimen from an individual to determine his or herhealth state. The size of the reference population depends on thecriteria used to select reference individuals. Depending on theselection criteria and particular application of the methods of theinvention, the reference population can be a relatively small number toa large number of individuals, including thousands of individuals.

The size of the reference population that is sufficient to determine ahealth-associated reference expression region for a group of moleculesdepends on the variability in expression of the molecules in thereference population and also on the degree of statistical separationfrom other reference populations. In some cases, the variability inexpression of the molecule in a population is due to genetic variationin the population. The greater the genetic variation, the larger thereference population is needed to provide a statistically usefulhealth-associated reference expression region. Accordingly, a smallerpopulation can be used if reference individuals have a similar geneticbackground. For example, the closest genetic relationship to anindividual is exhibited by an identical twin. It is therefore possibleto compare the expression levels of molecules in an individual to theexpression levels of the molecules in an identical twin of theindividual having appropriate reference criteria, for example, anidentical twin that is healthy. In such a case, an individual'sexpression profile can be compared to health-associated referenceexpression levels of molecules found in the identical twin to determinethe health state of the individual.

Beyond an identical twin, the individuals having the next closestgenetic similarity are family members who are blood relatives of anindividual for which determination of a comparative expression profileis desired. Thus, family members having appropriate reference criteriacan be used as a reference population. Due to the genetic similarity offamily members, a relatively small population can be used to determineuseful health-associated reference expression intervals, for example,populations of about 2 or more, about 3 or more, about 5 or more, about10 or more, about 20 or more, about 30 or more, about 50 or more, orabout 100 or more individuals. If relatively large populations ofrelated individuals are available, the populations can be about 200 ormore, about 300 or more, about 500 or more, about 1000 or more, about2000 or more, about 5000 or more, or even about 10,000 or moreindividuals. As described above, a reference population of familymembers is particularly useful for identifying molecules having suchvariability in expression that they are disregarded as molecules havingexpression levels correlated with the health state of an individual.

A reference population of family members can also be useful fordetermining polymorphic variations. For example, two unrelated familiescan be used as separate reference populations to determine expressionlevels of molecules. If desired, the family members can be selected sothat the family reference population is representative of a healthypopulation. A similar analysis can be performed on a geneticallyunrelated family. Thus, the expression levels of molecules in tworeference, healthy populations are determined. As described above, sucha population of related family members will exhibit less geneticvariability than a population of unrelated individuals. By comparing twogenetically unrelated but healthy reference populations to each other,molecules exhibiting variable expression between the two referencepopulations most likely represent genetic variability rather than adisease-specific variability. Such a comparison can be useful foridentifying those molecules exhibiting variability that is notassociated with a disease state, and such molecules can accordingly bedisregarded as molecules having expression levels correlated with thehealth state of an individual. Such a comparison, in combination with acomparison of reference health state and a disease state, can thus beused to identify sample molecules that are correlated with the healthstate of an individual.

Still another group of individuals having genetic similarity areindividuals in a particular ethnic group. Thus, a reference populationcan be selected from individuals in an ethnic group to determinehealth-associated reference expression intervals. Such a referencepopulation would include a larger population than a reference populationof family members since the genetic variation in an ethnic group wouldbe greater than in family members, for example, about 5 or more, about10 or more, about 20 or more, about 30 or more, about 50 or more, about100 or more, about 200 or more, about 500 or more, about 1000 or more,about 2000 or more, about 5000 or more, or even greater numbers ofindividuals. The expression levels of molecules in an individual of aparticular ethnic background can be compared to a health-associatedreference expression region determined for the ethnically relatedreference population. Using a health-associated reference expressionregion from a particular ethnic group can be desirable if theperturbation limit for one or more molecules expressed in that ethnicgroup lies within the health-associated reference expression intervalsof those molecules for the general population and if aberrant expressionof that molecule is associated with a disease in that ethnic group.

Still larger populations of reference individuals are used when thereference individuals are selected from the general population and arenot directed to a specific ethnic group. In such a case, the referenceindividuals can represent a relatively random sampling of a generalpopulation, which can include a sufficient number of individuals from asufficient variety of ethnic groups to be representative of the generalpopulation, for example, about 10 or more, about 20 or more, about 30 ormore, about 50 or more, about 100 or more, about 200 or more, about 500or more, about 1000 or more, about 2000 or more, about 5000 or more,about 10,000 or more, or even greater numbers of individuals. Selectionof a sufficient variety of ethnic groups allows the genetic variancebetween ethnic groups to be incorporated into the health-associatedreference expression region. Since a general population will havegreater genetic variation, a larger population of reference individualsis used to determine a health-associated reference expression region.The number and variety of ethnic groups to include in a generalreference population can be determined by one skilled in the artdepending on the ethnic diversity of test individuals for which acomparative expression profile is to be determined.

The use of a reference individual or reference population can also beapplied to identify a sample of molecules in a population of moleculesuseful for determining the health state of an individual. For example,if an expression profile of an individual having a disease is determinedand compared to a related family member or family members, the moleculeshaving differences in expression can include a sample of moleculesindicative of the health state of an individual. The expression levelsof those molecules exhibiting differences in expression can bedetermined in a reference population. For example, those moleculeshaving statistically useful health-associated reference expressionintervals represent a sample of molecules in a population of molecules.Accordingly, the sample of molecules can vary depending on theparticular disease, and disease-specific samples can be determined bycharacterizing the expression profile in individuals having a variety ofdiseases, if desired. For example, disease-specific samples of moleculescan be detected on directed targets containing corresponding ligandsthat bind to the sample of molecules.

Another method of identifying a sample of molecules useful forpredicting the health state of an individual is to pool groups ofreference individuals for comparison. Rather than individually measuringthe expression levels of molecules in each individual of a referencepopulation, specimens from reference individuals can be pooled. Forexample, healthy reference individuals can be pooled to generate ahealthy reference pool, and individuals having a particular disease canbe pooled separately from the healthy reference pool. The pooledreference populations can then be used to determine the expressionlevels of molecules in specimens from the pooled populations. Thedetermined expression levels of such a pooled population is essentiallyan average of the population. The “average” expression levels determinedfor the separate reference populations can be compared, and such acomparison is expected to reveal molecules having differentialexpression between the pooled samples. Such a set of differentiallyexpressed molecules can be used as a sample of molecules predictive ofthe health state of an individual having the disease of the pooleddisease reference population. Identification of disease-specificmolecules can be useful for identifying target ligands for a directedtarget. Such an analysis can be used for a dimensionality reduction, inwhich a smaller set of molecules is used as a predictor of the healthstate of an individual. Dimensionality reduction can therefore be usefulin identifying a sample of molecules predictive of the health state ofan individual.

Using a pool of reference individuals to identify a sample of moleculespredictive of the health state of an individual is useful forsimplifying the initial analysis and identification of a sample ofmolecules because it can provide a qualitative and quantitative analysisof the differential expression between two populations having differenthealth states without the need to perform assays on many individualsseparately. Essentially one assay can be performed for each pool ofreference populations rather than individual assays on each member ofthe population. Accordingly, if desired, large numbers of individualscan be pooled, including hundreds, thousands, or tens of thousands ofindividuals, including, about 10,000, about 20,000, about 30,000, about40,000 or even about 50,000 or more individuals, and convenientlyassayed as essentially one specimen.

In addition to pooling a reference population corresponding to aparticular disease, a pooled population can also be of individualsdiagnosed with a variety of diseases. Rather than identifying a sampleof molecules useful for diagnosing a particular disease, a pooledpopulation having a variety of diseases can be useful in a more generaldiagnostic assay for determining the health state of an individual sincea sample of molecules identified by such a pool would contain moleculesthat varied in expression in a variety of disease states.

Similarly, a pool of reference individuals having physiologicalperturbations can be pooled. Such physiological perturbations caninclude, for example, fasting, drug intake or drug withdrawal, exercise,and the like, as disclosed herein. Furthermore, a population ofindividuals having physiological perturbations can be pooled withdisease individuals to identify a more general set of sample moleculesuseful for determining a variety of health states. Such methods ofpooling disease and physiologically perturbed populations can be usefulfor identifying a sample of molecules and appropriate ligands foridentifying those molecules on a general target. The sample moleculesidentified by such pooled populations of disease and physiologicallyperturbed individuals can also include molecules whose relativeexpression changes, even though individual molecules are expressedwithin a healthy reference population, as disclosed herein (see FIG. 1).

Exemplary physiological perturbations include fasting, drug intake orwithdrawal, exercise, and the like. For example, physiologicalperturbations can include fasting, which is often used for lipidmeasurements or other physiological changes that are more immediatelyaffected by diet. Physiological perturbations can include a restingstate or sleep state, or can include exercise, for example, a stresstest or other form of physical exertion. A physiological perturbationcan also include the administration of safe compounds or drugs to testphysiological responses of an individual. For example, glucose tolerancecan be used to measure insulin response. Nitroglycerin can beadministered for vasodilation and to determine patient habituation. Anyof a variety of drugs or compounds that alter physiology but are knownto be safe and well tolerated by most individuals can be used tophysiologically perturb an individual and measure associated changes inexpression of molecules.

The methods of the invention can thus be used to diagnose disease statesor perturbed physiological states. The methods of the invention can alsobe used to identify changes in expression in response to drug treatment.Thus, by monitoring various populations of individuals, the methods ofthe invention can be used to predict the efficacy of a particular drugtreatment based on changes in expression of specimen molecules.Multidimensional multiparameter analysis is particularly useful foranalyzing more subtle changes in expression of molecules that can beassociated with the treatment of a disease.

A population of individuals sufficient to obtain a health-associatedreference expression interval for a general population of individualswould generally contain, tens, hundreds or thousands of referenceindividuals, depending on the method of determining expression levels aswell as the variability in expression of the sample of moleculesrepresentative of the health state of the reference population. Forexample, the population can contain, for example, about 20 or more,about 30 or more, about 50 or more, about 100 or more, about 200 ormore, about 500 or more or about 1000 or more individuals. A populationcan also contain about 2000 or more, about 3000 or more, about 4000 ormore, or even about 5000 or more individuals. Additionally, a populationcan contain about 7000 or more, about 10,000 or more, about 15,000 ormore or even about 20,000 or more individuals, depending on theparticular application. One skilled in the art can readily determine anappropriate sized population to determine a health-associated referenceexpression interval based on statistical analysis of the determinedreference expression ranges (see, for example, Solberg, supra, 1999).

Once a health-associated reference expression interval has been obtainedfor a sufficient number of molecules for a particular application, acomparative expression profile can be determined. A comparativeexpression profile is determined by comparing the expression levels of asample of molecules in a population of molecules to a health-associatedreference expression interval for each molecule. Such a comparativeexpression profile can be conveniently converted to a useful output, forexample, by assigning values as described above in aone-molecule-at-a-time analysis or by comparing the expression levels ofthe sample of molecules to one or more health-associated referenceexpression regions in a multidimensional analysis.

To determine the expression level of a molecule in an individual, aspecimen is obtained from the individual that is representative of theexpression level of molecules in the individual. A specimen can beobtained from an individual as a fluid or tissue specimen. For example,a tissue specimen can be obtained as a biopsy such as a skin biopsy,tissue biopsy or tumor biopsy. A fluid specimen can be blood, urine,saliva or other bodily fluids. A fluid specimen is particularly usefulin methods of the invention since fluid specimens are readily obtainedfrom an individual. Methods for collection of specimens are well knownto those skilled in the art (see, for example, Young and Bermes, inTietz Textbook of Clinical Chemistry, 3rd ed., Burtis and Ashwood, eds.,W.B. Saunders, Philadelphia, Chapter 2, pp. 42-72 (1999)). A specimencan optionally be fractionated into cell populations or subpopulations.A particularly useful method of fractionating a population of cells isto use ligands that bind to a cell surface molecule, for example,fluorescent antibodies that bind to a cell surface antigen followed byseparation in a fluorescence-activated cell sorter.

If desired, multiple specimens from an individual can be combined andanalyzed as a single specimen representative of the expression levels ofmolecules in an individual. Alternatively, multiple specimens from anindividual can be separately used to determine expression levels ofmolecules in the different specimens, and then the expression levelsfrom multiple specimens compared or averaged, so long as the specimensfrom the reference population are treated in the same manner or theexpression levels are correlated with appropriate controls and/orvalidation methods, as disclosed herein.

A specimen useful in methods of the invention contains one or moremolecules that are representative of the gene expression level and/orcellular expression level of molecules in the individual. Methods forobtaining specimens that preserve the expression profile of molecules ina specimen, including nucleic acids such as mRNA, polypeptides, smallmolecules, or post-translational modifications of such molecules, arewell known to those skilled in the art. Such methods include the use ofappropriate buffers and/or inhibitors, including nuclease, protease andphosphatase inhibitors, that preserve or minimize changes in theexpression level of molecules in the specimen. Such inhibitors include,for example, chelators such as ethylenediamine tetraacetic acid (EDTA),ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid(EGTA), protease inhibitors such as phenylmethylsulfonyl fluoride(PMSF), aprotinin, leupeptin, antipain and the like, and phosphataseinhibitors such as phosphate, sodium fluoride, vanadate and the like.Appropriate buffers and conditions for isolating molecules are wellknown to those skilled in the art and can be varied depending, forexample, on the type of molecule in the specimen to be characterizedwith respect to expression level (see, for example, Ausubel et al.,Current Protocols in Molecular Biology (Supplement 47), John Wiley &Sons, New York (1999); Harlow and Lane, Antibodies: A Laboratory Manual(Cold Spring Harbor Laboratory Press (1988); Harlow and Lane, UsingAntibodies: A Laboratory Manual, Cold Spring Harbor Press (1999); TietzTextbook of Clinical Chemistry, 3rd ed., Burtis and Ashwood, eds., W.B.Saunders, Philadelphia, (1999)).

If desired, the specimen can be incubated or processed in a manner toincrease the availability of molecules in the specimen for analyticalmethods disclosed herein, including binding to a target. For example, ifthe molecule to be detected in the specimen is a nucleic acid and thetarget ligand is a nucleic acid, the specimen can be incubated inbuffers and under conditions useful for preserving nucleic acids,particularly mRNA, and for detecting hybridization between nucleic acidmolecules. Such conditions are well known to those skilled in the art(Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., ColdSpring Harbor Press, Plainview, N.Y. (1989); Ausubel et al., CurrentProtocols in Molecular Biology (Supplement 47), John Wiley & Sons, NewYork (1999)). Furthermore, a specimen containing mRNA can be convertedto cDNA, if desired, using reverse transcriptase.

A specimen can also be processed to eliminate or minimize the presenceof interfering substances. For example, a specimen containing nucleicacids can be fractionated or extracted to remove potentially interferingnon-nucleic acid molecules. The specimen can also be treated to decreaseinterfering nucleic acids, for example, by treating a specimen withDNase or RNase to increase the ability to detect RNA or DNA,respectively. Various methods to fractionate a fluid specimen or cellextract are well known to those skilled in the art, includingsubcellular fractionation or chromatographic techniques such as ionexchange, hydrophobic and reverse phase, size exclusion, affinity,hydrophobic charge-induction chromatography, and the like (Ausubel etal., supra, 1999; Scopes, Protein Purification: Principles and Practice,third edition, Springer-Verlag, New York (1993); Burton and Harding, J.Chromatogr. A 814:71-81 (1998)).

If the molecule to be detected in the specimen is a polypeptide and thetarget ligand is an antibody, the specimen can be incubated in bufferssuitable for immunological detection methods, for example, the additionof detergents, including denaturants such as sodium dodecyl sulfate(SDS), if desired (Harlow and Lane, supra, 1988; Harlow and Lane, supra,1999). The specimen can also be fractionated, for example, into cellularor subcellular fractions, if desired.

Bodily fluid specimens are particularly useful in methods of theinvention due to ready availability. A particularly useful fluidspecimen is a blood specimen, particularly one containing leukocytes(WBCs). A specimen from an individual containing leukocytes isrepresentative of the physiological state of the individual and,therefore, is useful in determining the expression level of molecules inan individual that is indicative of the health state of the individual.Gene and cellular expression in leukocytes reflects many physiologicalsystems and states in the cell, for example, nervous, immune,cardiovascular, gastrointestinal, endocrine, hepatic, lymphatic,neuromuscular, renal, respiratory, skeletal, and urogenital systems.Pathologies in these systems and perturbations in organs of thesesystems are reflected in the leukocytes. Therefore, using an analyticalmethod that is useful for detecting molecules in a leukocyte specimenfrom an individual is particularly useful in methods of the invention.For example, a target that reflects expression of molecules inleukocytes, for example, a target containing leukocyte ESTs, can be usedto determine expression in leukocytes.

Furthermore, expression in leucocytes can be used to correlate changesin expression associated with other physiological systems such as thecardiovascular system, nervous system, or other systems, as disclosedherein. Since leukocytes reflect the physiological state in a variety ofsystems, leukocytes can be used as a specimen to determine whether thereis a change in the health state of the individual, including changes inone or more physiological systems associated with a disease or changesthat are associated with a physiological change such as drinking or drugintake, and the like.

Moreover, leukocytes can also be used to detect infectious disease dueto alterations in the physiological state of the individual. Inaddition, other physiological changes, including changes due toexercise, age, consumption of alcoholic beverages or intake of drugs arealso reflected in leukocytes. Thus, a specimen containing leukocytes isconvenient for determining an expression profile that can be correlatedwith the health state of an individual for a variety of physiologicalconditions, including exercise, age, drinking, and the like, in additionto disease states without the need for invasive biopsy procedures toobtain samples of tissues or organs that are directly involved in thedisease.

When using leukocytes as a specimen, a serum specimen from an individualcontaining leukocytes can be fractionated to isolate leukocytes, ifdesired, or subfractionated, for example, into macrophages, T cells, Bcells, granulocytes, monocytes, neutrophils, eosinophils, basophils,mast cells, and the like. Serum can be fractionated into a leukocytefraction or subfractionated using methods well known in clinicalchemistry and blood analysis. Leukocytes or subfractions thereof canalso be isolated by affinity binding methods specific for leukocytes orleukocyte subfractions. For example, an antibody binding step using aleukocyte-specific antibody can be used to isolate leukocytes. Theleukocytes can optionally be eluted from the affinity matrix, or thebound leukocytes can be directly used by lysing the leukocytes bound tothe affinity matrix. Similarly, antibodies specific for leukocytesubfractions such as T cell or B cell specific antibodies can be used tosubfractionate leukocytes. In addition, antibodies specific to cellsurface markers such as CD markers can be used to identify and/orisolate a subpopulation of cells. Such cell surface markers can also beused to determine the ratios of particular cell types in a specimen, forexample, using a cell sorting apparatus, which can also be an indicationof a disease state, a predisposition to developing a disease, or todetermine the outcome of a disease.

In one embodiment, a direct quantitation method is used to determine thelevel of expression of a molecule in a specimen. One such method is theisotope-coded affinity tag (ICAT) method (Gygi et al., NatureBiotechnol. 17:994-999 (1999) which is incorporated herein byreference). The ICAT method is particularly useful for proteomics-basedapplications. The ICAT method uses an affinity tag that can bedifferentially labeled with an isotope that is readily distinguishedusing mass spectrometry, for example, hydrogen and deuterium. The ICATaffinity reagent consists of three elements, an affinity tag, a linkerand a reactive group.

One element of the ICAT affinity reagent is an affinity tag that allowsisolation of peptides coupled to the affinity reagent by binding to acognate binding partner of the affinity tag. A particularly usefulaffinity tag is biotin, which binds with high affinity to its cognatebinding partner avidin, or related molecules such as streptavidin, andis therefore stable to further biochemical manipulations. Any affinitytag can be used so long as it provides sufficient binding affinity toits cognate binding partner to allow isolation of peptides coupled tothe ICAT affinity reagent.

A second element of the ICAT affinity reagent is a linker that canincorporate a stable isotope. The linker has a sufficient length toallow the reactive group to bind to a specimen polypeptide and theaffinity tag to bind to its cognate binding partner. The linker also hasan appropriate composition to allow incorporation of a stable isotope atone or more atoms. A particularly useful stable isotope pair is hydrogenand deuterium, which can be readily distinguished using massspectrometry as light and heavy forms, respectively. Any of a number ofisotopic atoms can be incorporated into the linker so long as the heavyand light forms can be distinguished using mass spectrometry. Exemplarylinkers include the 4,7,10-trioxa-1,13-tridecanediamine based linker andits related deuterated form,2,2′,3,3′,11,11′,12,12′-octadeutero-4,7,10-trioxa-1,13-tridecanediamine,described by Gygi et al. (supra, 1999). One skilled in the art canreadily determine any of a number of appropriate linkers useful in anICAT affinity reagent that satisfy the above-described criteria.

The third element of the ICAT affinity reagent is a reactive group,which can be covalently coupled to a polypeptide in a specimen. Any of avariety of reactive groups can be incorporated into an ICAT affinityreagent so long as the reactive group can be covalently coupled to aspecimen molecule. For example, a polypeptide can be coupled to the ICATaffinity reagent via a sulfhydryl reactive group, which can react withfree sulfhydryls of cysteine or reduced cysteines in a polypeptide. Anexemplary sulfhydryl reactive group includes an iodoacetamido group, asdescribed in Gygi et al. (supra, 1999). Other exemplary sulfhydrylreactive groups include maleimides, alkyl and aryl halides, α-haloacylsand pyridyl disulfides. If desired, the specimen polypeptides can bereduced prior to reacting with an ICAT affinity reagent, which isparticularly useful when the ICAT affinity reagent contains a sulfhydrylreactive group.

A reactive group can also react with amines such as Lys, for example,imidoesters and N-hydroxysuccinimidyl esters. A reactive group can alsoreact with carboxyl groups found in Asp or Glu, or the reactive groupcan react with other amino acids such as His, Tyr, Arg, and Met. Methodsfor modifying side chain amino acids in polypeptides are well known tothose skilled in the art (see, for example, Glazer et al., LaboratoryTechniques in Biochemistry and Molecular Biology: Chemical Modificationof Proteins, Chapter 3, pp. 68-120, Elsevier Biomedical Press, New York(1975); Pierce Catalog (1994), Pierce, Rockford Ill.). One skilled inthe art can readily determine conditions for modifying specimenmolecules by using various reagents, incubation conditions and time ofincubation to obtain conditions optimal for modification of specimenmolecule for use in methods of the invention.

The ICAT method is based on derivatizing a specimen molecule such as apolypeptide with an ICAT affinity reagent. A control reference specimenand a specimen from an individual to be tested are differentiallylabeled with the light and heavy forms of the ICAT affinity reagent. Thederivatized specimens are combined and the derivatized molecules cleavedto generate fragments. For example, a polypeptide molecule can beenzymatically cleaved with one or more proteases into peptide fragments.Exemplary proteases useful for cleaving polypeptides include trypsin,chymotrypsin, pepsin, papain, Staphylococcus aureus (V8) protease, andthe like. Polypeptides can also be cleaved chemically, for example,using CNBr or other chemical reagents.

Once cleaved into fragments, the tagged fragments derivatized with theICAT affinity reagent are isolated via the affinity tag, for example,biotinylated fragments can be isolated by binding to avidin in a solidphase or chromatographic format. If desired, the isolated, taggedfragments can be further fractionated using one or more alternativeseparation techniques, including ion exchange, reverse phase, sizeexclusion affinity chromatography and the like. For example, theisolated, tagged fragments can be fractionated by high performanceliquid chromatography (HPLC), including microcapillary HPLC.

The fragments are analyzed using mass spectrometry (MS). Because thespecimen molecules are differentially labeled with light and heavyaffinity tags, the peptide fragments can be distinguished on MS,allowing a side-by-side comparison of the relative amounts of eachpeptide fragment from the control reference and test specimens. Ifdesired, MS can also be used to sequence the corresponding labeledpeptides, allowing identification of molecules corresponding to thetagged peptide fragments.

An advantage of the ICAT method is that the pair of peptides tagged withlight and heavy ICAT reagents are chemically identical and thereforeserve as mutual internal standards for accurate quantification (Gygi etal., supra, 1999). Using MS, the ratios between the intensities of thelower and upper mass components of pairs of heavy- and light-taggedfragments provides an accurate measure of the relative abundance of thepeptide fragments. Furthermore, a short sequence of contiguous aminoacids, for example, 5-25 residues, contains sufficient information toidentify the unique polypeptide from which the peptide fragment wasderived (Gygi et al., supra, 1999). Thus, the ICAT method can beconveniently used to identify differentially expressed molecules, ifdesired.

The ICAT method can be used to quantitate the expression levels ofmolecules in reference individuals. Because the ICAT method is based ona direct comparison between two samples, the expression levels invarious reference individuals can be conveniently quantitated relativeto the same control reference specimen, for example, another referenceindividual or an appropriate cell line. Thus, the ICAT method can beconveniently used to quantitate the expression levels of molecules inreference individuals to determine a health-associated referenceexpression region.

Furthermore, the ICAT method can be used to quantitate the expressionlevels of molecules in an individual to be tested for his or her healthstate. The expression levels of molecules in a test individual can becompared to reference expression levels. For example, the referenceexpression levels can be those of a control reference specimen, whichare directly compared to the test individual using differential isotopelabeling in the ICAT method. The control reference specimen can be thesame as that used to determine the health-associated referenceexpression region. Alternatively, the control specimen can be differentthan that used to establish the health-associated reference expressionregion, so long as the expression levels of the control referencespecimen is correlated with the health-associated reference expressionregion.

The control reference specimen can also be a pool of referencespecimens. For example, the control reference specimen can be a pool oftwo or more specimens of reference individuals used to establish ahealth-associated reference region and can be a pool of all referenceindividuals, if desired. Such a pool of all reference individuals isexpected to result in a reference level that is essentially an averageof the reference individuals. One skilled in the art can readilydetermine a desired number of one or more reference individuals,including all reference individuals, to include in a pool for use as acontrol reference specimen. The amount of a pooled sample is adjustedaccordingly to allow direct comparison to the test individual, forexample, based on cell number, amount of protein, or some otherappropriate measure of the relative amount of control reference specimenand test specimen.

The above-described ICAT method can be performed as tandem MS/MS. A dualmode of MS can be performed in which MS alternates in successive scansbetween measuring relative quantities of peptides and recording ofsequence information of selected peptides (Gygi et al., supra, 1999).Other modes of MS include matrix-assisted laser desorption-time offlight (MALDI-TOF), an electrospray process with MS, and ion trap. Inion trap MS, fragments are ionized by electrospray and then put into anion trap. Trapped ions can then be separately analyzed by MS uponselective release from the ion trap. Fragments can also be generated inthe ion trap and analyzed.

In addition to polypeptides, the ICAT method can similarly be applied todetermining the expression level of nucleic acid molecules. In such acase, the ICAT affinity reagent incorporates a reactive group for anucleotide, for example, a group reactive with an amino group. The ICATaffinity reagent can incorporate functional groups specific for aparticular nucleotide or a nucleotide sequence of 2 or more nucleotides.The nucleic acid molecules can be cleaved enzymatically, for example,using one or more restriction enzymes, or chemically (see Sambrook etal., supra, 1989; Ausubel et al., supra, 1999).

In another embodiment, a binding assay is used to determine theexpression level of a specimen molecule. For example, molecules in aspecimen from the individual is contacted with a target. The targetcontains ligands, which can be essentially any type of molecule such aspolypeptide, nucleic acid, carbohydrate, lipid, or any organic derivedcompound, so long as the ligand can bind to a molecule that isrepresentative of the expression profile corresponding to one or moremolecules in a specimen. The choice of target ligand depends on whichtype of molecule in the specimen is to be detected.

For example, a target ligand useful for detecting nucleic acids such asmRNA in a specimen can be a nucleic acid. In such a target, the ligandsare representative of mRNAs expressed in a specimen and include ligandsthat can bind a sample of molecules predictive of the health state of anindividual. A target can contain nucleic acid ligands that arerepresentative of each mRNA in a specimen, or a target can containnucleic ligands that are representative of a subset of mRNAs expressedin a specimen so long as the number and representation of target ligandsare sufficient to generate an expression profile useful for determiningthe health state of an individual. A target containing nucleic acidligands representative of relatively low abundance or rare mRNAs in aspecimen is particularly useful when such low abundance mRNAs vary withthe health state of an individual. The number of ligands to include in atarget can be readily determined by one skilled in the art depending onthe particular application and number of specimen molecules desired tobe detected.

A target containing nucleic acid ligands allows determination of theexpression profile of nucleic acid molecules such as mRNA in a specimen.The nucleic acid ligands can be DNA or RNA and can be oligonucleotides.A target nucleic acid can also be peptide-nucleic acid molecules (PNA)having peptide and nucleic acid molecules covalently bound (Nielson,Current Opin. Biotechnol. 10:71-75 (1999)).

A target containing nucleic acid ligands can also be used to determinethe expression level of nucleic acid-binding polypeptides. Detection ofnucleic acid-binding polypeptides can be particularly useful if changesin expression of nucleic acid-binding polypeptides, for example,transcription factors, is associated with a disease or predisposition todeveloping a disease. Target nucleic acid ligands can additionally beaptamers that bind to specimen polypeptide molecules. Aptamers areoligonucleotides having binding affinity for polypeptides (Tuerk andGold, Science 249:505-510 (1990); Ellington and Szostak, Nature346:818-822 (1990); Joyce, Curr. Opin. Struct. Biol. 4:331-336 (1994);Gold et al., Annu. Rev. Biochem. 64:763-797 (1995); Jayasena, Clin.Chem. 45:1628-1650 (1999); Famulok and Mayer, Curr. Top. Microbiol.Immunol. 243:123-136 (1999)). A diversity of at least 10¹⁵ species canbe synthesized. For example, DNA aptamers can be synthesized withvariable nucleic acid sequences flanked on each end by recognition sitesfor PCR primers. If desired, aptamers that bind to a polypeptide can beselected and amplified, and such aptamers can have affinities greaterthan antibodies.

Nucleic acid ligands of the target are chosen based on the desiredspecimen molecules to be detected. For example, if a known subset ofspecimen nucleic acids is to be detected, the target nucleic acids cancorrespond to specific nucleic acid sequences that can hybridize to theknown subset of specimen nucleic acids. Similarly, if a known set ofnucleic acid-binding polypeptides is to be detected in the specimen, thenucleic acid ligands can be nucleic acid sequences that function asbinding sites for the nucleic acid-binding polypeptides. The targetnucleic acids for detecting nucleic acid-binding polypeptides can besingle stranded or double stranded depending on whether the nucleicacid-binding polypeptides bind to single or double stranded nucleicacids, for example, transcription factors that bind to double strandedDNA. Alternatively, the target nucleic acid ligands, eitherdouble-stranded or single stranded depending on the desired application,can be representative of expressed sequence tags (ESTs) corresponding toa particular cell type. For example, if the specimen from the individualis a leukocyte, the sequences of the target ligands can correspond tomRNAs representative of the expression pattern in a leukocyte. In such acase, the mRNAs of a specimen comprising a cell such as a leukocyte canbe known, or a target can contain mRNA sequences where each individualsequence is not necessarily known. Furthermore, a target containing ESTscan be analyzed with respect to the expression of particular mRNAs invarious physiological systems such as the cardiovascular system, thenervous system and the like. mRNAs expressed in particular systems canbe selected as potential sample molecules useful in determining changesin the health state of an individual affecting particular systems.

Additionally, the target nucleic acid ligands can be completely randomsequences such as random oligonucleotide sequences, which can begenerated by degenerate synthetic schemes. Random oligonucleotidesequences can be used as target ligands so long as the target contains asufficient number of random nucleotide sequences that are statisticallyrepresentative of a sufficient number of specimen molecules to provide auseful expression profile. One skilled in the art can readily selectappropriate nucleic acid ligands based on the particular application andthe specimen molecules to be detected so long as the target provides asufficient number of target ligands to determine an expression profileof an individual.

A target useful for detecting polypeptides in a specimen can containligands that specifically bind to the polypeptides. Target ligandsuseful for detecting polypeptides include nucleic acids, as describedabove, antibodies, peptides or small molecule ligands such as smallorganic molecules. Antibody ligands are particularly useful fordetecting polypeptides in a specimen, including various biochemicalforms of a polypeptide such as post-translational modifications and thepresence or absence of post-translational modifications. Antibodies canbe designed, for example, to detect the presence or absence ofphosphorylation at one or more sites of phosphorylation.

Methods for preparing antibodies for use as target ligands are wellknown to those skilled in the art. As used herein, the term “antibody”is used in its broadest sense to include polyclonal and monoclonalantibodies, as well as antigen binding fragments of such antibodies. Anantibody useful in the invention, or antigen binding fragment of such anantibody, is characterized by having specific binding activity for apolypeptide or a peptide portion thereof of at least about 1×10⁵ M⁻¹.Thus, Fab, F(ab′)₂ Fd, Fv, single chain Fv (scFv) fragments of anantibody and the like, which retain specific binding activity for apolypeptide, are included within the definition of an antibody. Specificbinding activity of an antibody for a polypeptide can be readilydetermined by one skilled in the art, for example, by comparing thebinding activity of an antibody to a particular polypeptide versus acontrol polypeptide that is not the particular polypeptide. Methods ofpreparing polyclonal or monoclonal antibodies are well known to thoseskilled in the art (see, for example, Harlow and Lane, Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory Press (1988)).

In addition, the term “antibody” as used herein includes naturallyoccurring antibodies as well as non-naturally occurring antibodies,including, for example, single chain antibodies, chimeric, bifunctionaland humanized antibodies, as well as antigen-binding fragments thereof.Such non-naturally occurring antibodies can be constructed using solidphase peptide synthesis, can be produced recombinantly or can beobtained, for example, by screening combinatorial libraries consistingof variable heavy chains and variable light chains as described by Huseet al. (Science 246:1275-1281 (1989)). These and other methods of makingfunctional antibodies are well known to those skilled in the art (Winterand Harris, Immunol. Today 14:243-246 (1993); Ward et al., Nature341:544-546 (1989) Harlow and Lane, supra, 1988); Hilyard et al.,Protein Engineering: A practical approach (IRL Press 1992); Borrabeck,Antibody Engineering, 2d ed. (Oxford University Press 1995)).

Antibody ligands useful in methods of the invention can be generatedhaving specificity for known specimen polypeptides, as described above.A particularly useful method for generating antibody ligands is based onusing combinatorial libraries consisting of variable heavy chains andvariable light chains (Huse et al., Science 246:1275-1281 (1989)). Theadvantage of using such a combinatorial antibody library is thatantibodies do not have to be individually generated for each specimenmolecule to be detected. No prior knowledge of the exact characteristicsof molecules in a specimen is required when using a combinatorialantibody library. All that is necessary is that a sufficient number ofantibody ligands be included in the target so that a representativenumber of specimen molecules can be detected and that a usefulexpression profile of an individual can be determined. If desired, anantibody library can be screened for binding to molecules expressed in aspecimen, for example, by selecting for antibodies that bind to specimenmolecules such as molecules expressed in leukocytes. The selectedantibodies can be used as target ligands for binding to specimenmolecules.

In addition to antibody ligands, organic molecule ligands, includingpeptides, can be used to detect molecules in a specimen. Such organicmolecule ligands can be conveniently generated using combinatorialchemistry methods. Methods for producing pluralities of compounds to useas target ligands, including chemical or biological molecules such assimple or complex organic molecules, metal-containing compounds,carbohydrates, peptides, proteins, peptidomimetics, glycoproteins,lipoproteins, nucleic acids, antibodies, and the like, are well known inthe art (see, for example, in Huse, U.S. Pat. No. 5,264,563; Francis etal., Curr. Opin. Chem. Biol. 2:422-428 (1998); Tietze et al., Curr.Biol., 2:363-371 (1998); Sofia, Mol. Divers. 3:75-94 (1998); Eichler etal., Med. Res. Rev. 15:481-496 (1995); Gordon et al., J. Med. Chem. 37:1233-1251 (1994); Gordon et al., J. Med. Chem. 37: 1385-1401 (1994);Gordon et al., Acc. Chem. Res. 29:144-154 (1996); Wilson and Czarnik,eds., Combinatorial Chemistry: Synthesis and Application, John Wiley &Sons, New York (1997)). When a library of peptides is used as ligandsfor detecting specimen polypeptides, the peptides can form intofunctional domains having binding activity to specimen polypeptides.Libraries containing large numbers of natural and synthetic compoundsalso can be obtained from commercial sources. Because a large number andvariety of ligands can be generated by such combinatorial methods, atarget containing organic molecule ligands can be readily prepared andused to determine the expression profile of an individual.

Target ligands can be attached to a solid support for contacting with aspecimen, or the target ligands can be in solution and contacted with aspecimen. Generally, target ligands are stably bound to a solid support,which can be a membrane such as a nylon or nitrocellulose membrane,glass, derivatized glass, silicon, plastic or other substrates. Thetarget molecules can be bound to a flat surface such as a membrane orplate or can be bound to spheres or beads. Alternatively, specimenmolecules can be bound to a solid support and contacted with targetligands in solution.

A convenient format for a target can be, for example, an arraycontaining a plurality of ligands such as nucleic acids, antibodies,peptides or small organic molecules. As used herein, an array refers toa format for presenting binding molecules where the ligands are stablybound to a solid support and arranged such that the binding to aparticular ligand on the array can be detected. An array format isparticularly convenient when a large number of molecules in a specimenis desired to be detected. For example, a target containing nucleic acidligands can be an array of random oligonucleotides or an array of ESTs.Such nucleic acid arrays can be purchased commercially or customsynthesized. Similarly, ligands such as antibodies, peptides or smallorganic molecules can be attached to a solid support in an array format.

The target ligands can be stably bound to a solid support via covalentinteractions or non-covalent interactions so long as the ligands remainbound to the solid support during incubation or wash steps required todetect specific binding of a specimen molecule to the target. Generally,target ligands are attached to a solid support, for example, throughcovalent bonds such as chemical crosslinks. A ligand can also bemodified with an affinity tag that facilitates binding and orcrosslinking of the ligand to the solid support. High affinitynon-covalent interactions such as those mediated by avidin andstreptavidin and the like can also be used to stably bind a ligand to asolid support.

It is understood that a target, as used herein, refers to the totalnumber of different ligands used to detect molecules in a specimen. Forexample, if the diversity of ligands required to determine theexpression profile of an individual requires the use of three individualarrays each containing different ligands, the target is considered to bethe ligands on all three arrays. Moreover, it is understood thatcontacting a specimen with a target contained on multiple arrays can beperformed simultaneously, sequentially, or even at different times, forexample, on different days or weeks or even months apart so long asappropriate conditions are used to allow comparison of the bindinginteractions, as described below.

The specimen is contacted with the target under conditions that allowspecific binding of the specimen molecules to the target ligands. Asused herein, specific binding means binding that is measurably differentfrom a non-specific interaction. Specific binding can be measured, forexample, by determining binding of a molecule compared to binding of acontrol molecule, which generally is a molecule of similar structurethat does not have binding activity, for example, a peptide of similarsize that lacks binding activity or a nucleic acid having a differentnucleotide sequence. Specificity of binding also can be determined, forexample, by competition with a control molecule, for example,competition with an excess of the same molecule. In this case, specificbinding is indicated if the binding of a molecule is competitivelyinhibited by itself.

The conditions for the contacting step of a specimen and target can varydepending on the particular type of specimen molecule and target ligand.The nature of the desired binding interaction between specimen moleculeand target ligand and the method used to detect specific binding is alsoconsidered when determining appropriate binding conditions. For example,if the specimen molecule is a nucleic acid and the target ligand is anucleic acid, the contacting step is carried out under conditions thatallow specific binding and detection of specific binding. Such methodsare well known to those skilled in the art, as described above (Sambrooket al., supra, 1989; Ausubel et al., supra, 1999). Typically, thebinding interaction between specimen nucleic acids and target nucleicacid ligands are carried out under conditions that allow specifichybridization between specimen molecules and target ligands. In such acase, the target ligands generally are single stranded nucleic acidmolecules that can hybridize to the specimen molecules. In contrast, ifthe specimen molecule is a nucleic acid-binding polypeptide such as atranscription factor and the target ligand is a nucleic acid, the targetligands can be double stranded nucleic acids since nucleic acid-bindingmolecules such as transcription factors often bind to double strandedDNA. One skilled in the art can readily determine the appropriatebiochemical form of target ligands, for example, single stranded ordouble stranded nucleic acid, and conditions for specific binding ofspecimen molecules depending on the particular binding interaction to bedetected.

The methods of the invention include the step of comparing theexpression levels of molecules in a specimen from an individual with ahealth-associated reference expression region. Although not required,the health-associated reference expression region for the molecules isgenerally determined prior to determining the expression levels ofmolecules in a specimen from a non-reference individual, that is, a testindividual. Furthermore, it is possible that the expression level of onemolecule in a specimen is determined at a different time than thedetermination of the expression level of a second molecule in aspecimen. Whether the expression level of a molecule is determinedsimultaneously with the determination of an expression level for asecond molecule in a specimen or the determination of ahealth-associated reference expression region of the molecules, it isunderstood that such determinations are made under conditions that allowa statistically useful comparison, even if obtained at different times.

One useful method to allow comparison between specimens analyzed atdifferent times is to use an internal control that can be used tonormalize results between specimens. A particularly useful internalcontrol can be, for example, a molecule in the specimen for which theexpression level does not significantly vary between a reference healthstate and a perturbed health state. An internal control molecule can bea molecule corresponding to or encoding molecules such as actin, othercytoskeletal proteins, or any polypeptide or encoding nucleic acid thatdoes not significantly vary between a reference health state or aperturbed health state such as a disease state. Alternatively or inaddition, an exogenous control molecule can be added to normalizevariability between specimens collected at different times or fromdifferent individuals.

The use of internal and exogenous controls allows determination of thereproducibility of specimen collection and analysis. One skilled in theart will know or can readily determine if the expression leveldetermined for a molecule, whether in a population of referenceindividuals for obtaining a health-associated reference expressionregion or in an individual for determining an individual expressionprofile, is reproducible and reliable for use in methods of theinvention based on statistical analysis and determination ofexperimental variability.

The binding of a specimen molecule to a target ligand can be detectedusing well known methods and is based on the particular type of specimenmolecule and target ligand binding interaction to be detected. Forexample, a specimen molecule or target ligand can be modified to includea detectable moiety, for example, a radiolabel, a fluorochrome, achromogen, a ferromagnetic substance, a luminescent tag, a detectablebinding agent such as biotin, an enzyme such as horse radish peroxidase(HRP), alkaline phosphatase, glucose oxidase, and the like, or otherdetectable moieties known in the art that are detectable by analyticalmethods. Methods suitable for detecting such moieties include, forexample, autoradiography or phosphorimaging, fluorescence spectroscopy,calorimetric detection, or light detection.

As used herein, a label refers to single atoms and molecules that areeither directly or indirectly involved in the production of a detectablesignal. Any label can be linked to target ligands or to specimenmolecules. These detectable atoms or molecules can be used alone or inconjunction with additional reagents. Such additional reagents arewell-known in clinical diagnostic chemistry. The linking of a label to asubstrate, for example, a specimen molecule or target ligand, includingnucleic acid, polypeptides, antibodies, and small organic molecules, iswell known in the art. For example, in the case of nucleic acids,nucleotides labeled with radioactive, fluorescent, or calorimetricmoieties can be incorporated enzymatically or chemically into a nucleicacid. In the case of specimen polypeptides, polypeptides can be modifiedby conjugating a detectable moiety with a chemical cross linking agentor metabolically labeling cells in a specimen to incorporate aradiolabel. As described above, an isotopic label such as an ICATaffinity reagent can also be conjugated to a specimen molecule anddetected by MS. Antibodies can be labeled by conjugating detectablelabels, including enzymes, using cross linking agents or, if theantibodies are expressed recombinantly, for example, using antibodylibraries, the antibodies can be labeled by expressing the antibodies asa fusion with a detectable peptide tag.

A method of detection that directly measures binding of a specimenmolecule to a target ligand can also be used. In such a case, thebinding of a specimen molecule to a target ligand is performed withouteither the specimen molecules or target ligands being directly labeled.Such indirect methods include using mass spectrometry or detectablesecondary reagents that bind to a specimen molecule or target ligand.

The choice of detection system will depend on the nature of the specimenmolecule and target ligand binding interaction. For example, a varietyof detection systems can be used if a specimen nucleic acid molecule isto be detected. Such methods include specific hybridization and/oramplification methods. Methods and conditions for hybridizing a specimennucleic acid molecule to a target nucleic acid ligand are well known tothose skilled in the art. Hybridization conditions can vary depending onthe stringency of the binding and washing conditions. Hybridizationreactions can be performed under low stringency, moderate stringency, orhigh stringency conditions. The conditions for various stringencyhybridization reactions are well known to those skilled in the art (seeSambrook et al., supra, 1989; Ausubel et al., supra, 1999).

The phrase stringent hybridization is used herein to refer to conditionsunder which polynucleic acid hybrids are stable. Typically, thehybridization reaction is performed under conditions of lowerstringency, followed by washes of varying, but higher, stringency.Reference to hybridization stringency relates to such washingconditions.

The phrase “moderately stringent hybridization” refers to conditionsthat permit target-nucleic acid to bind a complementary nucleic acid.The hybridized nucleic acids will generally have at least about 60′identity, at least about 75% identity, more at least about 85% identity;or at least about 90% identity. Moderately stringent conditions areconditions equivalent to hybridization in 50% formamide, 5×Denhart'ssolution, 5×SSPE, 0.2% SDS at 42° C., followed by washing in 0.2×SSPE,0.2% SDS, at 42° C.

High stringency hybridization refers to conditions that permithybridization of only those nucleic acid sequences that form stablehybrids in 0.018M NaCl at 65° C., for example, if a hybrid is not stablein 0.018M NaCl at 65° C., it will not be stable under high stringencyconditions, as contemplated herein. High stringency conditions can beprovided, for example, by hybridization in 50% formamide, 5× Denhart'ssolution, 5×SSPE, 0.2% SDS at 42° C., followed by washing in 0.1×SSPE,and 0.1% SDS at 65° C.

Low stringency hybridization refers to conditions equivalent tohybridization in 10 formamide, 5× Denhart's solution, 6×SSPE, 0.2% SDSat 22° C., followed by washing in 1×SSPE, 0.2% SDS, at 37° C. Denhart'ssolution contains 1% Ficoll, 1% polyvinylpyrolidone, and l % bovineserum albumin (BSA). 20×SSPE (sodium chloride, sodium phosphate,ethylene diamide tetraacetic acid (EDTA)) contains 3M sodium chloride,0.2M sodium phosphate, and 0.025 M (EDTA). Other suitable moderatestringency and high stringency hybridization buffers and conditions arewell known to those of skill in the art and are described, for example,in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed.,Cold Spring Harbor Press, Plainview, N.Y. (1989); and Ausubel et al.,supra, (1999).

If desired, a specimen nucleic acid can be amplified using methods suchas polymerase chain reaction (PCR). If the specimen nucleic acid is RNA,the RNA molecules can be reverse transcribed into cDNA. Methods ofamplifying nucleic acids by PCR and reverse transcription are well knownto those skilled in the art (see, for example, Dieffenbach and Dveksler,PCR Primer: A Laboratory Manual, Cold Spring Harbor Press (1995);Ausubel et al., supra, 1999).

To detect binding of a specimen nucleic acid molecule to a targetligand, the specimen molecules can be labeled with a detectable moietysuch as a radiolabel, fluorescent label, or calorimetric label. Whenspecimen mRNA is to be detected, a detectable moiety can beincorporated, for example, during reverse transcription of the mRNA intocDNA. Alternatively, the target ligand can be labeled to detect bindingof a specimen molecule, for example, the target ligand can be labeledwith a fluorescent label that is quenched upon binding of a nucleic acidmolecule. Another system is the molecular beacon system in which thetarget ligand contains a fluorescent label and a quencher such that afluorescent signal is emitted upon hybridization to a specimen molecule(Fang et al., J. Am. Chem. Soc. 121:2921-2922 (1999); Fang et al., SPIE,3602:149-155 (1999)). Furthermore, methods in which neither the specimenmolecule nor the target ligand is labeled can also be used to detect abinding interaction between a specimen nucleic acid molecule and targetnucleic acid ligand, for example, mass spectrometry.

Alternatively, a secondary reagent that is detectably labeled can beused to detect binding of nucleic acids. For example, a specimencontaining nucleic acid molecules can be hybridized to target nucleicacid ligands, and unbound specimen molecules can be removed. The targetcan then be contacted with secondary reagent nucleic acids containing adetectable moiety such as a radiolabel, fluorescent label, orcalorimetric label. Those target nucleic acid ligands that are bound tospecimen nucleic acid molecules are inaccessible to the labeledsecondary reagent nucleic acids whereas the secondary reagent can bindto unbound target nucleic acid ligands, allowing detection of bindinginteractions.

For detection of binding of a specimen polypeptide to a target antibodyligand, the detection methods can employ a labeled specimen polypeptide,a labeled target antibody ligand, or a labeled secondary reagent,similar to the methods described above for detecting nucleic acidbinding. For example, the specimen polypeptides can be metabolicallyradiolabeled, or a detectable moiety such as a radiolabel, fluorescent,or calorimetric label can be attached to specimen polypeptides byenzymatic or chemical means. Alternatively, a target antibody ligand canbe labeled, or binding of a target antibody ligand to a specimenmolecule can be detected using well known immunological detectionmethods (Harlow and Lane, supra, 1988; Harlow and Lane, supra, 1999).Methods of detecting binding of a target antibody ligand using wellknown immunological methods are particularly useful when the specimenmolecules are attached to a solid support.

Methods of detecting binding of a specimen polypeptide to a targetantibody ligand can also employ methods in which neither the specimenpolypeptide nor the target antibody ligand are detectably labeled, forexample, using mass spectrometry. Additionally, a labeled secondaryreagent can be used to detect binding interactions between a specimenpolypeptide and a target antibody ligand similar to the methodsdescribed above for detecting nucleic acid binding. For example, aspecimen containing polypeptide molecules can be contacted with targetantibody ligands, and unbound specimen molecules can be removed. Thetarget can then be contacted with labeled secondary reagents containinga detectable moiety such as a radiolabel, fluorescent, or calorimetriclabel and that can bind to unbound antibodies but not to antibodiesbound to specimen polypeptides. Those target antibody ligands that arebound to specimen polypeptide molecules are inaccessible to the labeledsecondary reagents whereas the labeled secondary reagents can bind tounbound target antibody ligands, allowing detection of bindinginteractions.

Detection of specimen polypeptide molecules bound to target nucleic acidligands can also be based on differential staining of polypeptides. Theuse of protein stains to detect binding of polypeptides to nucleic acidscan be particularly useful when detecting binding of polypeptides toaptamers. Alternatively, laser bombardment can be used to detect bindingof specimen polypeptides to target ligands.

The methods of the invention are based on determining the expressionlevels of molecules in a specimen or specimens to determine ahealth-associated reference expression region or to determine theexpression profile of an individual to compare to a health-associatedreference expression region. Therefore, the methods involve quantitationof the expression of molecules in a specimen. Methods for quantitativeassays of the expression level of a specimen molecule are well known tothose skilled in the art. For example, if desired, the target cancontain various amounts of a ligand to facilitate quantitation ofbinding of a specimen molecule.

Furthermore, a target can contain different amounts of target ligandssuitable for quantitating expression levels of specimen molecules basedon expected expression ranges of the specimen molecules. Such expectedranges can be determined using target-based methods, for example, usingarrays. Alternatively, quantitation of expression levels can beperformed by another method, for example, using a direct method such asICAT, and correlated with a target-based method such as an array. Thus,quantitation by a method such as ICAT can be used to establish expectedexpression ranges of molecules and to calibrate a target-based methodfor convenient use in an array format. Thus, the amount of differentligands on the target need not be identical and can be varied to provideoptimized detection of molecules in a specimen.

Methods for determining the levels of expression of small molecules arewell known to those skilled in the art. For example, methods ofanalyzing small molecules such as glucose, sugars, carbohydrates,sodium, potassium, chloride, calcium, chromium, iron, selenium,magnesium, manganese, molybdenum, zinc, copper, amino acids, lipids,neurotransmitters such as acetylcholine, dopamine, norepinephrine,epinephrine, seratonin, γ-aminobutyrate, and the like, as well as othersmall molecules disclosed herein, can be analyzed using well knownclinical chemistry methods (see, for example, Tietz Textbook of ClinicalChemistry, second edition, Burtis and Ashwood, eds., W.B. SaundersCompany, Philadelphia (1994); Tietz Textbook of Clinical Chemistry, 3rded., Burtis and Ashwood, eds., W.B. Saunders Co., Philadelphia (1999)).

The methods of the invention disclosed herein for detecting nucleicacids and/or polypeptides, particularly methods useful for detectinglarge numbers of molecules such as array-based methods, can be combinedwith well known methods of detecting expression levels of smallmolecules to determine the expression levels of more than one type ofmolecule. Exemplary methods of determining the levels of small moleculesinclude the use of enzyme-based assays, including calorimetric andradioenzymatic (incorporation of radioactive substrates), chromogenicassays, spectrophotometry, fluorescence spectroscopy, liquidchromatography, including ion exchange, affinity, HPLC, paperchromatography, gas chromatography, photometry atomic absorptionspectrometry, emission spectroscopy, including inductively coupledplasma emission spectroscopy, mass spectrometry, inductively coupledmass spectrometry, neutron activation analysis, X-ray fluorescencespectrometry, electrochemical techniques such as anodic strippingvoltametry, polarographic techniques, flame emission spectrophotometry,electrochemical methods such as ion selective electrodes, chemicaltitration, and the like (Tietz Textbook of Clinical Chemistry, secondedition, Burtis and Ashwood, eds., W.B. Saunders Company, Philadelphia(1994); Tietz Textbook of Clinical Chemistry, 3rd ed., Burtis andAshwood, eds., W.B. Saunders Co., Philadelphia (1999)). Small moleculeassay methods can also be adapted to accommodate multiple samples,including solid phase or array based formats.

Additional methods to those described above for measuring the expressionlevels of molecules in a sample can be used. Any new methods can becorrelated with a previously determined method that is useful fordetermining the expression levels of molecules in a sample. Once a dataset has been determined, for example, a sample of molecules correlatedwith a disease has been identified and a health-associated referenceexpression region has been determined by a particular method, thepreviously used method can be correlated with a new set of molecules ormethod of assaying the expression levels of the molecules. For example,the expression of molecules can be measured using the old method andcompared to a new method. By comparing the old and new methods using acalibration curve, the information determined by the old method can betransformed and correlated with a new method for measuring theexpression of molecules in a sample. The transformed method is validatedby correlating data derived by the two methods. If transformation doesnot provide a good correlation between the two methods, the new methodcan be validated by generating a new set of calibrations for the newmethod.

The methods of the invention described herein can also be used todiagnose a disease or condition in an individual. The invention thusprovides a method of diagnosing a disease. The method of diagnosing adisease can include the step of comparing the expression levels of asample of molecules in a population of molecules in a specimen from anindividual with health-associated reference expression intervals of themolecules in the sample, wherein an expression level within thehealth-associated reference expression intervals indicates a referencehealth state and wherein an expression level outside thehealth-associated reference expression interval indicates a diseasestate.

In addition, the method of diagnosing a disease can include the step ofdetermining a multidimensional coordinate point representative of theexpression levels of a sample of molecules in a population of moleculesin a specimen from the individual; comparing the multidimensionalcoordinate point to a health-associated reference expression region ofthe sample of molecules; and determining if the multidimensionalcoordinate point is within or outside the health-associated referenceexpression region, wherein the multidimensional coordinate point withinthe health-associated reference expression region indicates a referenceexpression profile and wherein the multidimensional coordinate pointoutside the health-associated reference expression region indicates aperturbed expression profile.

The methods can further include the step of determining the expressionlevels of a sample of molecules in a population of molecules in thespecimen. The method can also include the step of contacting a specimenfrom an individual with a target or directly comparing the expressionlevels of molecules with reference expression levels correlated with ahealth-associated reference expression region.

The methods of the invention can be used to determine the health stateof an individual and to diagnose a variety of diseases. The methods ofthe invention can be used to diagnose diseases, for example, cancer,including breast, prostate, ovarian, lung colorectal, hepatic, renal,leukemia, and lymphoma; cardiovascular diseases, including heartfailure, hypertension and atherosclerosis; respiratory diseases; renaldiseases; gastrointestinal diseases, including inflammatory boweldiseases such as Crohn's disease and ulcerative colitis; hepatic,gallbladder and bile duct diseases, including hepatitis and cirrhosis;hematologic diseases; metabolic diseases; endocrine and reproductivediseases, including diabetes; bone and bone mineral metabolism diseases;immune system diseases, including autoimmune diseases such as rheumatoidarthritis, lupus erythematosus, and other autoimmune diseases;musculoskeletal and connective tissue diseases, including arthritis;infectious diseases; and neurological diseases.

In addition to diagnosing various diseases, the methods of the inventioncan also be used to determine the health state of an individual as itrelates to the physiological state of the individual. For example, thehealth state of an individual can be determined to indicate if theindividual has consumed alcoholic beverages or drugs, has beenexercising, or other physiological changes that result in changes in theexpression profile of an individual relative to a reference population.

The invention additionally provides a method of diagnosing a healthstate in an individual. The method of diagnosing a health state caninclude the steps of determining the expression levels of a sample ofmolecules in a population of molecules in a specimen from an individual;comparing the expression levels with a health-associated referenceexpression region of the sample of molecules; and determining if theexpression levels of the sample of molecules is within or outside thehealth-associated reference expression region, wherein expression levelswithin the health-associated reference expression region indicates areference health state and wherein expression levels outside thehealth-associated reference expression region indicates a disease state.

The method of diagnosing a health state can also include the steps of(a) comparing the expression level of a molecule in a specimen from theindividual with a health-associated reference expression interval of themolecule; and (b) assigning a value of 0 if the expression level iswithin the health-associated reference expression interval or assigninga positive numerical value if the expression level is outside thehealth-associated reference expression interval, wherein an expressionlevel within the health-associated reference expression intervalindicates a reference health state and wherein an expression leveloutside the health-associated reference expression interval indicates aperturbed health state. Similarly, the expression levels can be comparedto one or more health-associated reference expression regions.

Also, the method of diagnosing a health state can includes the steps of(a) comparing the expression level of a molecule in a specimen from theindividual with a health-associated reference expression interval of themolecule; and (b) assigning a value of 0 if the expression level iswithin the health-associated reference expression interval, assigning apositive numerical value if the expression level is greater than thehealth-associated reference expression interval, or assigning a negativenumerical value if the expression level is less than thehealth-associated reference expression interval, wherein a value of 0indicates a reference health state and wherein a positive or negativenumerical value indicates a perturbed health state. Similar methods canbe performed comparing expression levels of a sample of molecules to ahealth-associated reference expression region.

In methods of the invention, the determination of the expressionlevel(s) of a molecule or group of molecules in a specimen allowscomparison to a health-associated reference expression interval for thatmolecule or to a health-associated reference expression region for thatgroup of molecules. Once the expression level(s) of a molecule or groupof molecules is determined, the expression level(s) can be inputted intoa method for comparing the expression level of the molecule to ahealth-associated reference expression interval or the expression levelsfor the group of molecules to a health-associated reference expressionregion. A value can be assigned based on whether the expression level ofthe molecule is within or outside a health-associated referenceexpression interval, particularly in a one-molecule-at-a-time analysis.Methods of comparing the expression level(s) of a molecule or group ofmolecules in a specimen to a health-associated reference expressionregion and optionally assigning a value based on whether the expressionlevel is within the health-associated reference expression region can beused to determine an expression profile based on the expression level ofa few molecules to a large number of molecules in a sample so long asthe number of molecules is sufficient to provide an expression profileof an individual that indicates the health state of the individual, andsuch information can be used to estimate the course of a disease.

The methods of the invention can be conveniently performed on a computerapparatus. Any of the methods or particular steps of the methodsdisclosed herein can be performed on a computer apparatus. Performingone or more steps of an invention method on a computer apparatus isparticularly useful when analyzing a large number of parameters such asa large number of sample molecules.

The invention thus provides a computer apparatus comprising a processor;main memory in communication with the processor; and a comparativeexpression profiler in communication with the main memory configured tocarrying out the computer-executed steps of (a) comparing the expressionlevel of a molecule with a health-associated reference expressioninterval of the molecule; and (b) assigning a value of 0 if theexpression level is within the health-associated reference expressioninterval or assigning a positive numerical value if the expression levelis outside the health-associated reference expression interval, whereinan expression level within the health-associated reference expressioninterval indicates a reference expression profile and wherein anexpression level outside the health-associated reference expressioninterval indicates a perturbed expression profile.

The invention also provides a computer apparatus comprising a processor;main memory in communication with the processor; and a comparativeexpression profiler in communication with the main memory configured tocarrying out the computer-executed steps of (a) comparing the expressionlevel of a molecule with a health-associated reference expressioninterval of the molecule; and (b) assigning a value of 0 if theexpression level is within the health-associated reference expressioninterval, assigning a positive numerical value if the expression levelis greater than the health-associated reference expression interval, orassigning a negative numerical value if the expression level is lessthan the health-associated reference expression interval, wherein anexpression level within the health-associated reference expressioninterval indicates a reference expression profile and wherein anexpression level outside the health-associated reference expressioninterval indicates a perturbed expression profile (see FIG. 1). In acomputer apparatus of the invention, steps (a) and (b) can be repeatedone or more times, particularly in a one-molecule-at-a-time analysis.

The invention provides a computer apparatus, comprising a processor;main memory in communication with the processor; and a comparativeexpression profiler in communication with the main memory configured tocarrying out the computer-executed steps of: (a) determining amultidimensional coordinate point representative of the expressionlevels of a sample of molecules from an individual; (b) comparing themultidimensional coordinate point with a health-associated referenceexpression region, wherein the multidimensional coordinate point withinthe health-associated reference expression region indicates a referenceexpression profile and wherein the multidimensional coordinate pointoutside the health-associated reference expression region indicates aperturbed expression profile (see FIG. 7).

An invention computer apparatus can further be configured to carry outthe computer-executed step of determining the expression level of themolecule. It is understood that any of the methods disclosed herein thatare conveniently performed on a computer apparatus can be included assteps to be performed by an invention computer apparatus. For example, acomputer based method can be used to select a sample of molecules in apopulation of molecules in a specimen by determining which moleculeshave a health-associated reference region that is statistically usefulor to perform any of the statistical methods, as disclosed herein. Acomputer apparatus is also useful for determining a multidimensionalcoordinate point and comparing the coordinate point to ahealth-associated reference expression region. A molecule that does nothave a statistically reproducible health-associated reference expressioninterval in a reference population can be excluded from the samplemolecules by the computer based method.

The methods of the invention directed to assigning values based onwhether the expression level of a molecule is within or outside ahealth-associated reference expression interval can be advantageouslyperformed using a computer apparatus since the methods are directed toassigning numerical values, which can be readily processed on a computerapparatus. The use of a computer apparatus is also convenient since ahealth-associated reference expression interval for a large number ofmolecules can be conveniently stored and accessed for comparison to theexpression level of a molecule from a specimen. Similarly, the methodsof the invention directed to determining a multidimensional coordinatepoint and comparing to a health-associated reference expression regioncan be conveniently performed on a computer apparatus, and the computerapparatus can be used to store instructions for determining inclusion inone or more health-associated reference expression regions of variousreference populations as well as a database of health-associatedreference regions for comparison to a test individual.

It is understood that a computer apparatus of the invention need notitself store the health-associated reference expression interval ofvarious molecules or a health-associated reference expression region.The computer apparatus contains a comparative expression profiler, whichis capable of comparing an expression level of a molecule to ahealth-associated reference expression interval or expression levels fora group of molecules to a health-associated reference expression region.However, a database containing health-associated reference expressionintervals, health-associated reference expression regions, orinstructions for determining inclusion in the regions can beconveniently accessed using appropriate hardware, software, and/ornetworking, for example, using hardware interfaced with networks,including the internet.

By using various hardware, software and network combinations, themethods of the invention can be conveniently performed in a variety ofconfigurations. For example, a single computer apparatus can contain acomparative expression profiler, a database containing a collection ofhealth-associated reference expression intervals for one or moremolecules or one or more health-associated reference expression regions,and instructions for determining inclusion in one or morehealth-associated reference expression regions. Alternatively, thecomputer apparatus can contain a comparative expression profiler whilethe database of health-associated reference expression intervals orhealth-associated reference expression regions is stored on a separatemedium. In addition, instructions for inclusion in one or morehealth-associated reference expression regions can be contained on aseparate computer apparatus or separate medium, or combined with thecomputer apparatus containing the comparative expression profiler or thedatabase on a separate medium. Such a separate medium can be anothercomputer apparatus, a storage medium such as a floppy disk, Zip disk ora server such as a file-server, which can be accessed by a carrier wavesuch as an electromagnetic carrier wave. Thus, a computer apparatuscontaining a comparative expression profiler can remotely access adatabase, for example, a database stored on a file-server and accessiblevia a network such as the internet. One skilled in the art will know orcan readily determine appropriate hardware, software or networkinterfaces that allow interconnection of an invention computerapparatus.

The invention also provides an apparatus comprising a comparativeexpression profiler and a means for determining the expression level ofa molecule. Such a determining means can include a device whichprocesses a specimen from an individual using the methods disclosedherein for determining the expression level of a molecule in a specimen.Such a device is one that can carry out the steps of contacting aspecimen with a target and determining the expression level of aspecimen molecule. The integration of a determining means with acomparative expression profiler in a single apparatus is particularlyuseful when a specimen is to be processed in a single location such as adiagnostic laboratory or physician's office.

A determining means and computer apparatus containing a comparativeexpression profiler can also be separate devices that are convenientlyinterfaced. For example, separate devices can be interfaced via atransportable medium, for example, a floppy disk, Zip disk, magneticdisk, external hard disk, and the like, which can be convenientlytransferred from one device to the another. Alternatively, separatedevices can be interfaced via a network. A network connection can be aphysical linkage between the devices via a cable connection or can beconnected via a carrier wave using any convenient combination of cables,servers, nodes, and the like, including connections via the internet ora similar network.

The use of separate devices for a determining means and a comparativeexpression profiler is particular useful for network applications thatcan be conveniently performed at a remote site. For example, adetermining means can be a simple kit that contains an array of targetligands and appropriate buffers and reagents for processing a specimento detect specimen molecules. Such a kit can be used, for example, in aclinical laboratory, a hospital, a physician's office, an ambulance, oreven in the privacy of an individual's home.

Any of the methods, or portions thereof, disclosed herein can be adaptedto a kit format for use in a remote location separate from a comparativeexpression profiler. For example, after exposing a component to aspecimen in a remote location, the kit component exposed to the specimencan be forwarded to a clinical laboratory for analysis and determinationof expression levels. Alternatively, the kit can contain componentssufficient for determining the expression levels of specimen moleculesat the remote location. After determining the expression levels ofmolecules in a specimen at a remote location, the information can beinterfaced with a comparative expression profiler at a differentlocation.

In the case of remote determination of expression levels, a simpleinterface between the determining means and a comparative expressionprofiler can be via a home or office computer. A convenient method toinput expression levels from the determining means to a computerapparatus containing a comparative expression profiler can be by placingthe determining means on a scanner, scanning the determining means arrayto convert the expression level of bound specimen molecules to anelectronic output, and sending the scanned expression level informationto a computer apparatus containing a comparative expression profiler viaa network such as the internet. Using a scanner to detect expressionlevels of specimen molecules is particularly useful when the method ofdetection is a calorimetric signal. However, it is understood that anydetection method suitable for detecting a specimen molecule can beadapted for remote use in a clinical laboratory, physician's office, orindividual's home. For example, a hand held device incorporatingsuitable micro-detection systems, small scale assays, and other suitablemethods for assaying samples on a small scale can optionally be used inremote detection of a specimen.

The invention further provides a computer-readable medium having storedthereon a plurality of sequences of instructions, the plurality ofsequences of instructions including sequences of instructions which,when executed by a processor, cause the processor to perform the stepsdescribed above for execution on a computer apparatus. It is understoodthat any of the methods disclosed herein can be provided as an inventioncomputer-readable medium. The invention additionally provides a carrierwave carrying instructions for a processor, the instructions which, whenexecuted by the processor, cause the processor to perform the stepsdescribed above for execution on a computer apparatus. It is understoodthat any of the methods disclosed herein can be provided as an inventioncarrier wave.

Referring to FIG. 6, a flow diagram that depicts the computer-executedsteps of an embodiment of the invention is shown. Step 100 starts theimplementation of an embodiment of the invention. In step 110, theexpression level of a molecule is compared to a health-associatedreference expression interval for that molecule. In step 120, a value of0 is assigned if the expression level of the molecule is within thehealth-associated reference expression interval. In step 130, a positivenumerical value is assigned if the expression level of the molecule isgreater than a health-associated reference expression interval for thatmolecule. In step 140, a negative numerical value is assigned if theexpression level of the molecule is less than a health-associatedreference expression interval.

In step 150, an inquiry is performed to determine if there is a moleculehaving an expression level that is not assigned a value. If the answeris “yes,” then step 110 is repeated for the molecule having anexpression level that is not assigned a value. If the answer is “no,” aninquiry is performed at step 160 to determine if any of the assignedvalues are non-zero values. If the answer is “no,” a normal expressionprofile is indicated in step 170. If the answer is “yes,” the positivevalues are summed to generate a positive summation value in step 180. Instep 190, the negative values are summed to generate a negativesummation value. In step 200, a perturbed expression profile isindicated. The method steps of determining a comparative expressionprofile end in step 210.

Referring to FIG. 7, a flow diagram that depicts the computer-executedsteps of an embodiment of the invention is shown. Step 300 starts theimplementation of an embodiment of the invention. In step 310, amultidimensional coordinate point representative of the expressionlevels of the sample of molecules is determined. In step 320, themultidimensional coordinate point is compared to a health-associatedreference expression region. In step 330, an inquiry is performed todetermine if the multidimensional coordinate point is within thehealth-associated reference expression region. If the answer is “yes,” areference expression profile is indicated in step 340. If the answer is“no,” a perturbed expression profile is indicated in step 350. Themethod steps of determining a comparative expression profile end in step360. The expression levels of a sample of molecules determinedseparately can be inputted to determine a multidimensional coordinatepoint representative of the expression levels of the sample ofmolecules.

Referring to FIG. 8, a block diagram of computer system 10, which can beemployed to implement the present invention, is shown. Computer system10 has operating system 15, processor 20, main memory 30, comparativeexpression profiler 40, display screen 50, input device 60, media drive70, disk storage 80, and output device 90, each of which is connected tosystem unit 10. Operating system 15 is an operating system such as UNIX,MS-DOS, Windows, or OS. The processor 20 is a general purposeprogrammable processor such as an Intel PENTIUM processor or a Motorolaprocessor, suitable for a mid-size personal computer such as DEC, IBM,Macintosh and the like. The main memory 30 can be well known randomaccess memory (RAM) that is sufficiently large to hold the necessaryprogramming and data structures. The comparative expression profiler 40in communication with main memory carries out computer-executable steps.For example, the comparative expression profiler can carry out thecomputer executable steps of comparing the expression level of amolecule with a health-associated reference expression interval for themolecule; and assigning a numerical value if the expression is within oroutside a health-associated reference expression interval. The computerexpression profiler can also carry out the computer executable steps ofdetermining a multidimensional coordinate point representative of theexpression levels of a sample of molecules from an individual; andcomparing the multidimensional coordinate point with a health-associatedreference expression region, wherein the multidimensional coordinatepoint within the health-associated reference expression region indicatesa reference expression profile and wherein the multidimensionalcoordinate point outside the health-associated reference expressionregion indicates a perturbed expression profile.

The display screen 50 is a screen for visualizing, for example, inputdata. The input device 60 is a mouse or a keyboard, or a combinationthereof, or any other device to input information. The media drive 70 isa drive, such as a tape drive, a disk drive or a CD drive, that providesthe computer system 10 access to the comparative expression profiler 40.The disk storage 80 is a device, such as a floppy disk, magnetic tape,Zip disk, external hard drive and the like that provides storagecapacity for data. The output device 90 is a device such as a modem orportal that allows interfacing with a network.

It is understood that modifications which do not substantially affectthe activity of the various embodiments of this invention are alsoprovided within the definition of the invention provided herein.Accordingly, the following examples are intended to illustrate but notlimit the present invention.

EXAMPLE I Calculation Methodology Using Multivariate ClassificationTheory

This example describes a calculation methodology using multivariateclassification theory to classify health-associated regions ofmultidimensional space.

Data are available on expression levels corresponding to a set ofmolecules for individuals with known health states, for example,healthy, ovarian cancer, prostate cancer, diabetes, and the like. Thenumber m corresponds to the number of different health states. Thecalculation steps involved are: (1) estimate the probabilitydistribution of the observed data vector for each health state; (2)estimate the costs of misclassification for each combination of healthstates; (3) estimate the a priori probabilities of a random individualbeing a member of each health state; and (4) determine the optimalcalculation to be performed when classifying a new individual. Thedevelopment given here is based upon multivariate statistical methodssuch as those of T. W. Anderson (An Introduction to MultivariateStatistical Analysis, Second Edition, Wiley, New York, 1984, Section6.7).

(1) Estimation of the Probability Distribution of the Data for a GivenHealth State

The estimated probability density function for a vector x of molecularexpression levels for health state i is denoted by p_(i) (x). Manymethods are available for this purpose. For example, a model can assumethat the distribution is multivariate normal and use the sample averageexpression level for each molecule (averaged over individuals known tobe in the given health state) and the sample covariance matrix ofexpression levels as estimates of the mean vector and covariance matrixof the multivariate normal distribution specifying the data distributionfor this health state. Exploratory data analysis can be used todetermine whether the multivariate normal assumption is appropriate.Alternatives such as mixture distributions, multivariate tdistributions, transformation or kernel smoothing techniques can also beused.

(2) Estimation of the Costs of Misclassification

Costs are denoted C(j|i) representing the cost of misclassifying anindividual as health state j when he or she actually is in health statei (where i,j=1, . . . , m). Complete flexibility is allowed in thesetting of relative costs of misclassification in that a different costfigure can be set for each combination of health states. Thus, the costof misclassifying a healthy individual as cancerous can be set either tobe the same or different from the cost of classifying a cancerousindividual as healthy. With m health states, costs can be specified form(m−1) combinations of health states. One available choice of costs isto set them all equal to 1, which says that any and allmisclassifications are equally costly.

(3) Estimation of the a Priori Probabilities of Health States

Epidemiological data on the incidence of each disease in the generalpopulation or a specific population can be used to estimate these apriori probabilities for the health states, which will be denoted q₁,q₂, . . . , q_(m).

(4) The Optimal Calculation for Classifying a New Individual

In order to minimize the expected cost, averaged over many individualsclassified by the system, the optimal decision rule is as follows. A newindividual with expression levels specified by a vector x for a set ofmolecules is classified as being in health state k if

${{\sum\limits_{\underset{i \neq k}{i = 1}}^{m}\; {q_{i}{p_{i}(x)}{C( {ki} )}}} < {\sum\limits_{\underset{i \neq j}{i = 1}}^{m}\; {q_{i}{p_{i}(x)}{C( {ji} )}}}},\; {j = 1},\ldots \mspace{14mu},\mspace{11mu} m,{j \neq k}$

This is the calculation that determines the health-associated referenceregion containing the vector x.

For the data set shown in FIG. 3 for three health states and twomolecular expression levels, the resulting classification regions areshown under the assumptions that each of the three populations isbivariate normal, the costs of misclassification are all equal, and theprior probabilities are 0.7, 0.2, and 0.1 for the three groups. Becausehealth state 3 is rare, the optimal classification scheme reverts to themore common (and more disperse) health s and 2 at the upper left.

This example demonstrates that a statistical classification method canbe applied to multiple parameters in a two-dimensional analysis toclassify three distinct health states corresponding to health-associatedreference regions for three populations of individuals.

EXAMPLE II Logistic Regression Analysis

This example describes the analysis of a data set for three healthstates and two molecular expression levels using logistic regressionanalysis.

The data set was created starting with pseudorandom computer-generatednumbers and then applying a different mathematical transformation foreach health related reference group. For the data set shown in FIG. 4Afor three health states and two molecular expression levels, theresulting classification regions are shown using logistic regressionanalysis under the assumptions that the costs of misclassification areall equal, and the prior probabilities are 0.2, 0.5, and 0.3 for thethree groups. Because health state 2 is the most common in thepopulation, the classification tends to favor this group at the upperright where data are sparse.

The classification regions are based on three separate logisticregression analyses, one to predict each health state, where eachanalysis used the molecular expression levels for all health states butcoded the independent variable to indicate the health state to bepredicted. To allow for the curvature in the data, the predictorvariables were chosen to be cubic polynomials in the predictor variableswith a backward stepwise selection process to omit terms that do notcontribute to the prediction. The resulting predicted probability foreach health state can be scaled by its prior probability of occurrencein the population, and the resulting scores compared. The health statewith the largest score is the chosen classification, while the relativevalues of all three scores indicate the relative likelihoods of thethree health states.

The assignment of new individuals “A” and “B” to one of the threedefined health states were determined. The molecular expression levelsof two new individuals “A” and “B”, with unknown health states, areshown in FIG. 4B, with A indicated as “×” and B indicated as “+.”

The following method was used for computing the degree of confidence inthe assignment of a new individual: (a) compute the predictedprobability for each health state using the results of the logisticregression analyses (where these results do not include the newindividual) evaluated at the expression levels for the new individual;(b) multiply each of these numbers by the prior probability of thathealth state occurring in the population; (c) divide each of the threeresulting numbers by their sum in order to convert them intoprobabilities that add up to 1. The results of these steps are therelative probabilities that the new individual belongs to each healthgroup.

The degree of confidence in the assignment of individual A to healthgroup 1 was assessed by examining the relative probabilities ofindividual A belonging to each health group, and the results wereconsistent with FIG. 4B, which shows that individual A is clearlywell-described as being within the data for individuals with healthstate 1. The results show that individual A has a 97.0% chance of beingin health state 1, a 2.8% chance of being in health state 2, and a 0.2%chance of being in health state 3, as predicted using the model.

Individual B was also assigned to a health state, although the degree ofconfidence was less than for individual A. The degree of confidence inthe assignment of individual B to health group 2 was assessed, and theresults are consistent with FIG. 4B, which shows that individual B isnear the boundary that separates individuals with health state 2 fromthose having health state 3. The results show that individual B has a2.1% chance of being in health state 1, a 74.2% chance of being inhealth state 2, and a 23.60 chance of being in health state 3, aspredicted using the model.

This example shows that logistic regression analysis can be usedclassify the health states of a group of reference individuals and theassignment of an individual to a reference health state.

EXAMPLE III Machine Learning by Boosting of Individual Molecules

This example describes classification analysis using a machine learningalgorithm called “boosting” to combine a chosen group of simpleone-molecule-at-a-time decision rules to obtain an effective healthclassification.

The data set was created starting with pseudorandom computer-generatednumbers and then applying a different mathematical transformation foreach health related reference group. For the data set shown in FIG. 5for three health states and two molecular expression levels, theresulting classification regions are shown for a machine-learningtechnique that uses boosting to combine several one-molecule-at-a-timeanalyses to form a classification region under the assumption that theprior probabilities are 0.6, 0.3, and 0.1 for the three groups. In thiscase, 8 boosting steps have been taken. The method used here is based onthe AdaBoost.M1 algorithm described by Freund and Schapire (J. Computerand System Sciences, 55:119-139 (1997)).

The boosting technique in machine learning generally relies on a set ofsimple “weak learners” that are trained on the data with successiveweightings to give more importance to initial misclassifications in aneffort to improve the results. By selecting a set of weak learners andletting them vote on the most likely classification, the boostingtechnique is able to create a consensus decision rule that is muchstronger than any individual weak learner.

In this example, the weak learners are simple decision rules based onone-molecule-at-a-time analysis in which a molecule is chosen (in thiscase, either molecule 1 or molecule 2), and then two threshold values aand b are chosen with a≦b. An ordering of the health states 1, 2, and 3,which can be permuted, is also specified, perhaps 2, 3, 1. The decisionrule corresponding to these threshold values and this ordering of healthstates would decide on health state 2 if the molecular expression levelof this molecule is less than or equal to a, would decide on healthstate 3 if the molecular expression level is between a and b, and woulddecide health state 1 if the expression level is at least b.

Once the weak learners have been specified, the AdaBoost.M1 algorithm(Freund and Schapire, supra, 1997) operates automatically, as follows.

-   -   (a) Define weights w(i) to represent the initial, prior        probabilities for the given data indexed by i.    -   (b) Loop as t goes from 1 to T, where T is the number of        boosting iterations to be used.        -   (b.1) Define probabilities p(i) equal to w(i) divided by the            sum of the w(i) so that p(i)=w(i)/Σw(j). Note that these            weights and probabilities will change as the algorithm            proceeds.        -   (b.2) Find the optimal weak learner that minimizes the            expected error rate with respect to the current            probabilities, where the error rate of a weak learner is            defined as the sum of the p(i) for those observations that            are misclassified by the weak learner.        -   (b.3) If this optimal weak learner has an error rate larger            than 0.5, then set t equal to t−1 and stop.        -   (b.4) Define β(t)=bestError/(1=bestError) using the error            rate for the optimal weak learner using the error rate            calculation as specified in step (b.2), that is, bestError            refers to the error rate computed using the definition of            step (b.2).        -   (b.5) Update the weights by replacing w(i) with β(t)×w(i)            for those observations i that were classified correctly.            This has the effect of downweighting those observations that            were correctly classified.    -   (c) Weak learners t have now been selected, where t=T, unless        the method is stopped early due to error rate >0.5.    -   (d) To assign a classification to a new observation, first note        that each of the t selected weak learners assigns a health state        to the new observation, although individual weak learners can        assign different health states. These t weak learners are        allowed to vote, giving weak learner k weight ln(1/β(k)). The        health state receiving the largest total weight from the        selected weak learners is the assigned classification.

This example shows that machine learning by boosting of individualmolecules can be used classify the health states of a group of referenceindividuals.

Throughout this application various publications have been referenced.The disclosures of these publications in their entireties are herebyincorporated by reference in this application in order to more fullydescribe the state of the art to which this invention pertains.

Although the invention has been described above, it should be understoodthat various modifications can be made without departing from the spiritof the invention. Accordingly, the invention is limited only by theclaims.

1-16. (canceled)
 17. A method of predicting a drug response in anindividual, comprising: (a) determining a multidimensional coordinatepoint representative of the expression levels of a sample of moleculesin a specimen from an individual treated with a drug; (b) comparing saidmultidimensional coordinate point to a drug response-associatedreference expression region for individuals treated with said drug; and(c) determining if said multidimensional coordinate point for saidindividual is within or outside said drug response-associated referenceexpression region, wherein said multidimensional coordinate point withinsaid drug response-associated reference expression region indicates saidindividual has a substantially similar response to said drug asindividuals in a drug response reference population used for said drugresponse-associated reference expression region.
 18. The method of claim17, further comprising the step of inputting the expression level ofsaid molecules in said sample.
 19. The method of claim 17, furthercomprising the step of determining the expression level of saidmolecules in said sample.
 20. The method of claim 19, wherein theexpression levels of said sample of molecules in said specimen aredetermined by direct comparison with reference expression levelscorrelated with health-associated reference expression intervals of saidmolecules in said sample.
 21. The method of claim 17, further comprisingthe step of contacting said specimen with a target.
 22. The method ofclaim 17, wherein said specimen is selected from the group consisting ofleukocytes, blood, and serum.
 23. The method of claim 21, wherein saidtarget is an array.
 24. The method of claim 17, wherein said moleculesin said specimen comprise nucleic acids.
 25. The method of claim 21,wherein said target comprises nucleic acid ligands.
 26. The method ofclaim 17, wherein said molecules in said specimen comprise polypeptides.27. The method of claim 21, wherein said target comprises antibodyligands.
 28. The method of claim 17, wherein said molecules in saidspecimen comprise small molecules.
 29. A method of predicting a drugresponse in an individual, comprising: (a) determining amultidimensional coordinate point representative of the expressionlevels of a sample of molecules in a specimen comprising leukocytes froman individual treated with a drug; (b) comparing said multidimensionalcoordinate point to a drug response-associated reference expressionregion for individuals treated with said drug; and (c) determining ifsaid multidimensional coordinate point for said individual is within oroutside said drug response-associated reference expression region,wherein said multidimensional coordinate point within said drugresponse-associated reference expression region indicates saidindividual has a substantially similar response to said drug asindividuals in a drug response reference population used for said drugresponse-associated reference expression region.
 30. A method ofcategorizing drug responsiveness in a population, comprising: (a)determining a multidimensional coordinate point representative of theexpression levels of a sample of molecules in specimens from apopulation of individuals treated with a drug; (b) identifying a firstgroup of individuals having a substantially similar response to saiddrug; and (c) determining a drug response-associated referenceexpression region of said first group of individuals using saidmultidimensional coordinate points of said first group of individuals,thereby categorizing the drug responsiveness of said first group ofindividuals.
 31. The method of claim 30, further comprising the stepsof: (d) identifying a second group of individuals having a substantiallysimilar response to said drug, said drug response in said second groupbeing different than the drug response of said first group; and (e)determining a drug response-associated reference expression region ofsaid second group of individuals using said multidimensional coordinatepoints of said second group of individuals, thereby categorizing thedrug responsiveness of said second group of individuals.
 32. The methodof claim 31, further comprising optionally repeating steps (d) and (e)one or more times for an additional group of individuals having asubstantially similar response to said drug, said drug response in saidadditional group of individuals being different than the drug responseof identified groups.
 33. The method of claim 30, further comprising thestep of inputting the expression level of said molecules in said sample.34. The method of claim 30, further comprising the step of determiningthe expression level of said molecules in said sample.
 35. The method ofclaim 34, wherein the expression levels of said sample of molecules insaid specimen are determined by direct comparison with referenceexpression levels correlated with health-associated reference expressionintervals of said molecules in said sample.
 36. The method of claim 30,further comprising the step of contacting said specimen with a target.37. The method of claim 30, wherein said specimen is selected from thegroup consisting of leukocytes, blood, and serum.
 38. The method ofclaim 36, wherein said target is an array.
 39. The method of claim 30,wherein said molecules in said specimen comprise nucleic acids.
 40. Themethod of claim 36, wherein said target comprises nucleic acid ligands.41. The method of claim 30, wherein said molecules in said specimencomprise polypeptides.
 42. The method of claim 36, wherein said targetcomprises antibody ligands.
 43. The method of claim 30, wherein saidmolecules in said specimen comprise small molecules.